Antibodies against PAK5

ABSTRACT

The present invention relates to the nucleic acid molecules encoding an STE20-related family of novel protein kinases, ZC1, ZC2, ZC3, ZC4, STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, KHS2, SULU1, SULU3, GEK2, PAK4 and PAK5, segments and domains thereof, as well as various methods useful for the diagnosis and treatment of various kinase-related diseases and conditions. Mammalian nucleic acid molecules encoding these kinases are particularly disclosed, and more specifically human sources of these nucleic acids are disclosed.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/688,188filed Oct. 16, 2000, which is a divisional of U.S. application Ser. No.09/291,417, filed Apr. 13, 1999, which in turn claims priority to U.S.Provisional Application Ser. No. 60/081,784, filed Apr. 14, 1998. Thisapplication claims only subject matter disclosed in the parentapplication and therefore presents no new matter.

The instant application contains a “lengthy” Sequence Listing which hasbeen submitted via triplicate CD-R in lieu of a printed paper copy, andis hereby incorporated by reference in its entirety. Said CD-R arelabeled “CRF”, “Copy 1” and “Copy 2”, respectively, and each containsonly one identical 329 Kb file (38602329.APP).

FIELD OF THE INVENTION

The present invention relates to novel kinase polypeptides, nucleotidesequences encoding the novel kinase polypeptides, as well as variousproducts and methods useful for the diagnosis and treatment of variouskinase-related diseases and conditions.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedto aid in understanding the invention, but is not admitted to be or todescribe prior art to the invention.

Cellular signal transduction is a fundamental mechanism whereby externalstimuli that regulate diverse cellular processes are relayed to theinterior of cells. One of the key biochemical mechanisms of signaltransduction involves the reversible phosphorylation of proteins, whichenables regulation of the activity of mature proteins by altering theirstructure and function.

The best characterized protein kinases in eukaryotes phosphorylateproteins on the hydroxyl moiety of serine, threonine and tyrosineresidues. These kinases largely fall into two groups, those specific forphosphorylating serines and threonines, and those specific forphosphorylating tyrosines. Some kinases, referred to as “dualspecificity” kinases, are able to phosphorylate on tyrosine as well asserine/threonine residues.

Protein kinases can also be characterized by their location within thecell. Some kinases are transmembrane receptor-type proteins capable ofdirectly altering their catalytic activity in response to the externalenvironment such as the binding of a ligand. Others arenon-receptor-type proteins lacking any transmembrane domain. They can befound in a variety of cellular compartments from the inner surface ofthe cell membrane to the nucleus.

Many kinases are involved in regulatory cascades wherein theirsubstrates may include other kinases whose activities are regulated bytheir phosphorylation state. Ultimately the activity of some downstreameffector is modulated by phosphorylation resulting from activation ofsuch a pathway.

Protein kinases are one of the largest families of eukaryotic proteinswith several hundred known members. These proteins share a 250-300 aminoacid domain that can be subdivided into 12 distinct subdomains thatcomprise the common catalytic core structure. These conserved proteinmotifs have recently been exploited using PCR-based cloning strategiesleading to a significant expansion of the known kinases.

Multiple alignment of the sequences in the catalytic domain of proteinkinases and subsequent parsimony analysis permits the segregation ofrelated kinases into distinct branches or subfamilies including:tyrosine kinases, cyclic-nucleotide-dependent kinases,calcium/calmodulin kinases, cyclin-dependent kinases and MAP-kinases,serine-threonine kinase receptors, and several other less definedsubfamilies.

SUMMARY OF THE INVENTION

Through the use of a targeted PCR cloning strategy and of a “motifextraction” bioinformatics script, mammalian members of the STE20-kinasefamily have been identified as part of the present invention. Multiplealignment and parsimony analysis of the catalytic domain of all of theseSTE20-family members reveals that these proteins cluster into 9 distinctsubgroups. Classification in this manner has proven highly accurate notonly in predicting motifs present in the remaining non-catalytic portionof each protein, but also in their regulation, substrates, and signalingpathways. The present invention includes the partial or completesequence of new members of the STE20-family, their classification,predicted or deduced protein structure, and a strategy for elucidatingtheir biologic and therapeutic relevance.

Thus, a first aspect of the invention features an isolated, enriched, orpurified nucleic acid molecule encoding a kinase polypeptide selectedfrom the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7,ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.

By “isolated” in reference to nucleic acid is meant a polymer ofnucleotides conjugated to each other, including DNA and RNA, that isisolated from a natural source or that is synthesized. The isolatednucleic acid of the present invention is unique in the sense that it isnot found in a pure or separated state in nature. Use of the term“isolated” indicates that a naturally occurring sequence has beenremoved from its normal cellular (i.e., chromosomal) environment. Thus,the sequence may be in a cell-free solution or placed in a differentcellular environment. The term does not imply that the sequence is theonly nucleotide chain present, but that it is essentially free (about90-95% pure at least) of non-nucleotide material naturally associatedwith it, and thus is distinguished from isolated chromosomes.

By the use of the term “enriched” in reference to nucleic acid is meantthat the specific DNA or RNA sequence constitutes a significantly higherfraction (2-5 fold) of the total DNA or RNA present in the cells orsolution of interest than in normal or diseased cells or in the cellsfrom which the sequence was taken. This could be caused by a person bypreferential reduction in the amount of other DNA or RNA present, or bya preferential increase in the amount of the specific DNA or RNAsequence, or by a combination of the two. However, it should be notedthat enriched does not imply that there are no other DNA or RNAsequences present, just that the relative amount of the sequence ofinterest has been significantly increased. The term “significant” isused to indicate that the level of increase is useful to the personmaking such an increase, and generally means an increase relative toother nucleic acids of about at least 2 fold, more preferably at least 5to 10 fold or even more. The term also does not imply that there is noDNA or RNA from other sources. The other source DNA may, for example,comprise DNA from a yeast or bacterial genome, or a cloning vector suchas pUC19. This term distinguishes from naturally occurring events, suchas viral infection, or tumor type growths, in which the level of onemRNA may be naturally increased relative to other species of mRNA. Thatis, the term is meant to cover only those situations in which a personhas intervened to elevate the proportion of the desired nucleic acid.

It is also advantageous for some purposes that a nucleotide sequence bein purified form. The term “purified” in reference to nucleic acid doesnot require absolute purity (such as a homogeneous preparation).Instead, it represents an indication that the sequence is relativelymore pure than in the natural environment (compared to the natural levelthis level should be at least 2-5 fold greater, e.g., in terms ofmg/mL). Individual clones isolated from a cDNA library may be purifiedto electrophoretic homogeneity. The claimed DNA molecules obtained fromthese clones could be obtained directly from total DNA or from totalRNA. The cDNA clones are not naturally occurring, but rather arepreferably obtained via manipulation of a partially purified naturallyoccurring substance (messenger RNA). The construction of a cDNA libraryfrom mRNA involves the creation of a synthetic substance (cDNA) and pureindividual cDNA clones can be isolated from the synthetic library byclonal selection of the cells carrying the cDNA library. Thus, theprocess which includes the construction of a cDNA library from mRNA andisolation of distinct cDNA clones yields an approximately 10⁶-foldpurification of the native message. Thus, purification of at least oneorder of magnitude, preferably two or three orders, and more preferablyfour or five orders of magnitude is expressly contemplated.

By a “kinase polypeptide” is meant 32 (preferably 40, more preferably45, most preferably 55) or more contiguous amino acids set forth in theamino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or thecorresponding full-length amino acid sequence; 250 (preferably 255, morepreferably 260, most preferably 270) or more contiguous amino acids setforth in the amino acid sequence SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, or SEQ ID NO:105, or the corresponding full-length amino acidsequence; 27 (preferably 30, more preferably 40, most preferably 45) ormore contiguous amino acids set forth in the amino acid sequence SEQ IDNO:18; 16 (preferably 20, more preferably 25, most preferably 35) ormore contiguous amino acids set forth in the amino acid sequence SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, or SEQ IDNO:103 or the corresponding full-length amino acid sequence; 6(preferably 10, more preferably 15, most preferably 25) or morecontiguous amino acids set forth in the amino acid sequence of SEQ IDNO:97 or SEQ ID NO:99, 22 (preferably 30, more preferably 35, mostpreferably 45) or more contiguous amino acids set forth in the aminoacid sequence of SEQ ID NO:101, or the corresponding full-length aminoacid sequence; 78 (preferably 80, more preferably 85, most preferably90) or more contiguous amino acids set forth in the amino acid sequenceSEQ ID NO:107 or functional derivatives thereof as described herein. Forsequences for which the full-length sequence is not given, the remainingsequences can be determined using methods well-known to those in the artand are intended to be included in the invention. In certain aspects,polypeptides of 100, 200, 300 or more amino acids are preferred. Thekinase polypeptide can be encoded by a full-length nucleic acid sequenceor any portion of the full-length nucleic acid sequence, so long as afunctional activity of the polypeptide is retained, not to includefragments containing only amino acids 1-22 of SEQ ID NO:13 or only aminoacids 1-33 of SEQ ID NO:107.

The amino acid sequence will be substantially similar to the sequenceshown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:100, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or thecorresponding full-length amino acid sequence, or fragments thereof, notto include fragments consisting only of the amino acid sequences 1-22 ofSEQ ID NO:13 or 1-33 of SEQ ID NO:107. A sequence that is substantiallysimilar to the sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107will preferably have at least 90% identity (more preferably at least 95%and most preferably 99-100%) to the sequence of SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, or SEQ ID NO:107.

By “identity” is meant a property of sequences that measures theirsimilarity or relationship. Identity is measured by dividing the numberof identical residues by the total number of residues and gaps andmultiplying the product by 100. “Gaps” are spaces in an alignment thatare the result of additons or deletions of amino acids. Thus, two copiesof exactly the same sequence have 100% identity, but sequences that areless highly conserved, and have deletions, additions, or replacements,may have a lower degree of identity. Those skilled in the art willrecognize that several computer programs are available for determiningsequence identity using standard parameters, for example Blast(Altschul, et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2(Altschul, et al. (1990) J. mol. biol. 215: 403-410), and Smith-Waterman(Smith, et al. (1981) J. Mol. Biol. 147: 195-197).

In preferred embodiments, the invention features isolated, enriched, orpurified nucleic acid molecules encoding a kinase polypeptide comprisinga nucleotide sequence that: (a) encodes a polypeptide having the aminoacid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107;(b) is the complement of the nucleotide sequence of (a); (c) hybridizesunder highly stringent conditions to the nucleotide molecule of (a) andencodes a naturally occurring kinase polypeptide; (d) encodes a kinasepolypeptide having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO: 103, SEQ ID NO:105, or SEQ ID NO:107,except that it lacks one or more, but not all, of the following segmentsof amino acid residues: 1-21, 22-274, or 275-416 of SEQ ID NO:5, 1-31,32-308, 309-489 or 490-516 of SEQ ID NO:6, 1-178 or 179-414 of SEQ IDNO:7, 1-22, 23-289, 290-526, 527-640, 641-896, or 897-1239 of SEQ IDNO:13, 1-255, 256-442, 443-626, 627-954, or 955-1297 of SEQ ID NO:14,1-255, 256-476, 477-680, 681-983, or 984-1326 of SEQ ID NO:15, 1-13,14-273, 274-346, 347-534, or 535-894 of SEQ ID NO:18, 1-21, 22-277,278-427, 428-637, 638-751, or 752-898 of SEQ ID NO:22, 1-66, 67-215,216-425, 426-539, 540-786, or 787-887 of SEQ ID NO:23, 1-25, 26-273,274-422, 423-632, or 633-748 of SEQ ID NO:24, 1-51, 52-224, 225-393,394-658, or 659-681 of SEQ ID NO:29, 1-25, 26-281, 284-430, 431-640,641-754, 755-901, or 902-1001 of SEQ ID NO:31, 1-10, 11-321, or 322-373of SEQ ID NO:97, 1-57, 58-369, or 370-418 of SEQ ID NO:99, 1-52, 53-173,174-307, 308-572, or 573-591 of SEQ ID NO:103, 1-24, 25-289, 290-397,398-628, 629-872, or 873-1227 of SEQ ID NO:105, or 1-33, 34-294,295-337, 338-472, 473-724, or 725-968 of SEQ ID NO:107; (e) is thecomplement of the nucleotide sequence of (d); (f) encodes a polypeptidehaving the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31; SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107 fromamino acid residues 1-21, 22-274, or 275-416 of SEQ ID NO:5, 1-31,32-308, 309-489, or 490-516 of SEQ ID NO:6, 1-178 or 179-414 of SEQ IDNO:7, 23-289, 290-526, 527-640, 641-896, or 897-1239 of SEQ ID NO:13,1-255, 256-442, 443-626, 627-954, or 955-1297 of SEQ ID NO:14, 1-255,256-476, 477-680, 681-983, or 984-1326 of SEQ ID NO:15, 1-13, 14-273,274-346, 347-534, or 535-894 of SEQ ID NO:18, 1-21, 22-277, 278-427,428-637, 638-751, or 752-898 of SEQ ID NO:22, 1-66, 67-215, 216-425,426-539, 540-786, or 787-887 of SEQ ID NO:23, 1-25, 26-273, 274-422,423-632, or 633-748 of SEQ ID NO:24, 1-51, 52-224, 225-393, 394-658, or659-681 of SEQ ID NO:29, 1-25, 26-281, 282-430, 431-640, 641-754,755-901, or 902-1001 of SEQ ID NO:31, 1-10, 11-321, or 322-373 of SEQ IDNO:97, 1-57, 58-369, or 370-418 of SEQ ID NO:99, 1-52, 53-173, 174-307,308-572, or 573-591 of SEQ ID NO:103, 1-24, 25-289, 290-397, 398-628,629-872, or 873-1227 of SEQ ID NO:105, or 1-33, 34-294, 295-337,338-472, 473-724, or 725-968 of SEQ ID NO:107; (g) is the complement ofthe nucleotide sequence of (f); (h) encodes a polypeptide having theamino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97,SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ IDNO:107, except that it lacks one or more of the domains selected fromthe group consisting of a N-terminal domain, a catalytic domain, aC-terminal domain, a coiled-coil structure region, a proline-richregion, a spacer region, an insert, and a C-terminal tail; or (i) is thecomplement of the nucleotide sequence of (h).

The term “complement” refers to two nucleotides that can form multiplefavorable interactions with one another. For example, adenine iscomplementary to thymine as they can form two hydrogen bonds. Similarly,guanine and cytosine are complementary since they can form threehydrogen bonds. A nucleotide sequence is the complement of anothernucleotide sequence if all of the nucleotides of the first sequence arecomplementary to all of the nucleotides of the second sequence.

The term “domain” refers to a region of a polypeptide which contains aparticular function. For instance, N-terminal or C-terminal domains ofsignal transduction proteins can serve functions including, but notlimited to, binding molecules that localize the signal transductionmolecule to different regions of the cell or binding other signalingmolecules directly responsible for propagating a particular cellularsignal. Some domains can be expressed separately from the rest of theprotein and function by themselves, while others must remain part of theintact protein to retain function. The latter are termed functionalregions of proteins and also relate to domains.

The term “N-terminal domain” refers to the extracatalytic region locatedbetween the initiator methionine and the catalytic domain of the proteinkinase. The N-terminal domain can be identified following aSmith-Waterman alignment of the protein sequence against thenon-redundant protein database to define the N-terminal boundary of thecatalytic domain. Depending on its length, the N-terminal domain may ormay not play a regulatory role in kinase function. An example of aprotein kinase whose N-terminal domain has been shown to play aregulatory role is PAK65, which contains a CRIB motif used for Cdc42 andrac binding (Burbelo, P. D. et al. (1995) J. Biol. Chem. 270,29071-290740).

The N-terminal domain spans amino acid residues 1-21 of the sequence setforth in SEQ ID NO:5, amino acid residues 1-31 of the sequence set forthin SEQ ID NO:6, amino acid residues 1-22 of the sequence set forth inSEQ ID NO:13, amino acid residues 1-13 of the sequence set forth in SEQID NO:18, amino acid residues 1-21 of the sequence set forth in SEQ IDNO:22, amino acid residues 1-25 of the sequence set forth in SEQ IDNO:24, amino acid residues 1-51 of the sequence set forth in SEQ IDNO:29, amino acid residues 1-25 of the sequence set forth in SEQ IDNO:31, amino acid residues 1-57 of the sequence set forth in SEQ IDNO:99, amino acid residues 1-52 of the sequence set forth in SEQ IDNO:103, amino acid residues 1-24 of the sequence set forth in SEQ IDNO:105, or amino acid residues 1-33 of the sequence set forth in SEQ IDNO:107.

The term “catalytic domain” refers to a region of the protein kinasethat is typically 25-300 amino acids long and is responsible forcarrying out the phosphate transfer reaction from a high-energyphosphate donor molecule such as ATP or GTP to itself(autophosphorylation) or to other proteins (exogenous phosphorylation).The catalytic domain of protein kinases is made up of 12 subdomains thatcontain highly conserved amino acid residues, and are responsible forproper polypeptide folding and for catalysis. The catalytic domain canbe identified following a Smith-Waterman alignment of the proteinsequence against the non-redundant protein database.

The catalytic domain spans amino acid residues 22-274 of the sequenceset forth in SEQ ID NO:5, residues 32-308 of the sequence set forth inSEQ ID NO:6, residues 1-178 of the sequence set forth in SEQ ID NO:7,residues 23-289 of the sequence set forth in SEQ ID NO:13, residues1-255 of the sequence set forth in SEQ ID NO:14, residues 1-255 of thesequence set forth in SEQ ID NO:15, residues 14-273 of the sequence setforth in SEQ ID NO:18, residues 22-277 of the sequence set forth in SEQID NO:22, residues 1-66 of the sequence set forth in SEQ ID NO:23,residues 26-273 of the sequence set forth in SEQ ID NO:24, residues394-658 of the sequence set forth in SEQ ID NO:29, residues 26-281 ofthe sequence set forth in SEQ ID NO:31, residues 1-278 of the sequenceset forth in SEQ ID NO:97, residues 58-369 of the sequence set forth inSEQ ID NO:99, residues 1-103 of the sequence set forth in SEQ ID NO:101,residues 308-572 of the sequence set forth in SEQ ID NO:103, residues25-289 of the sequence set forth in SEQ ID NO:105, or residues 34-294 ofthe sequence set forth in SEQ ID NO:107.

The term “catalytic activity”, as used herein, defines the rate at whicha kinase catalytic domain phosphorylates a substrate. Catalytic activitycan be measured, for example, by determining the amount of a substrateconverted to a phosphorylated product as a function of time. Catalyticactivity can be measured by methods of the invention by holding timeconstant and determining the concentration of a phosphorylated substrateafter a fixed period of time. Phosphorylation of a substrate occurs atthe active-site of a protein kinase. The active-site is normally acavity in which the substrate binds to the protein kinase and isphosphorylated.

The term “substrate” as used herein refers to a molecule phosphorylatedby a kinase of the invention. Kinases phosphorylate substrates onserine/threonine or tyrosine amino acids. The molecule may be anotherprotein or a polypeptide.

The term “C-terminal domain” refers to the region located between thecatalytic domain or the last (located closest to the C-terminus)functional domain and the carboxy-terminal amino acid residue of theprotein kinase. By “functional” domain is meant any region of thepolypeptide that may play a regulatory or catalytic role as predictedfrom amino acid sequence homology to other proteins or by the presenceof amino acid sequences that may give rise to specific structuralconformations (i.e. coiled-coils). The C-terminal domain can beidentified by using a Smith-Waterman alignment of the protein sequenceagainst the non-redundant protein database to define the C-terminalboundary of the catalytic domain or of any functional C-terminalextracatalytic domain. Depending on its length and amino acidcomposition, the C-terminal domain may or may not play a regulatory rolein kinase function. An example of a protein kinase whose C-terminaldomain may play a regulatory role is PAK3 which contains aheterotrimeric Gb subunit-binding site near its C-terminus (Leeuw, T. etal (1998) Nature, 391, 191-195).

The C-terminal domain spans amino acid residues 275-416 of the sequenceset forth in SEQ ID NO:5, residues 309-489 of the sequence set forth inSEQ ID NO:6, residues 179-414 of the sequence set forth in SEQ ID NO:7,residues 897-1239 of the sequence set forth in SEQ ID NO:13, residues955-1297 of the sequence set forth in SEQ ID NO:14, residues 984-1326 ofthe sequence set forth in SEQ ID NO:15, residues 535-894 of the sequenceset forth in SEQ ID NO:18, residues 752-898 of the sequence set forth inSEQ ID NO:22, residues 279-330 of the sequence set forth in SEQ IDNO:97, residues 370-418 of the sequence set forth in SEQ ID NO:99, orresidues 873-1227 of the sequence set forth in SEQ ID NO:105.

The term “signal transduction pathway” refers to the molecules thatpropagate an extracellular signal through the cell membrane to become anintracellular signal. This signal can then stimulate a cellularresponse. The polypeptide molecules involved in signal transductionprocesses are typically receptor and non-receptor protein tyrosinekinases, receptor and non-receptor protein phosphatases, SRC homology 2and 3 domains, phosphotyrosine binding proteins (SRC homology 2 (SH2)and phosphotyrosine binding (PTB and PH) domain containing proteins),proline-rich binding proteins (SH3 domain containing proteins),nucleotide exchange factors, and transcription factors.

The term “coiled-coil structure region” as used herein, refers to apolypeptide sequence that has a high probability of adopting acoiled-coil structure as predicted by computer algorithms such as COILS(Lupas, A. (1996) Meth. Enzymology 266: 513-525). Coiled-coils areformed by two or three amphipathic □-helices in parallel. Coiled-coilscan bind to coiled-coil domains of other polypeptides resulting in homo-or heterodimers (Lupas, A. (1991) Science 252: 1162-1164).Coiled-coil-dependent oligomerization has been shown to be necessary forprotein function including catalytic activity of serine/threoninekinases (Roe, J. et al. (1997) J. Biol. Chem. 272: 5838-5845).

The coiled-coil structure region spans amino acid residues 290-526 ofthe sequence set forth in SEQ ID NO:13, residues 256-442 of the sequenceset forth in SEQ ID NO:14, residues 256-476 of the sequence set forth inSEQ ID NO:15, residues 428-637 of the sequence set forth in SEQ IDNO:22, residues 216-425 or 540-786 of the sequence set forth in SEQ IDNO:23, residues 423-632 of the sequence set forth in SEQ ID NO:24,residues 431-640 or 755-901 of the sequence set forth in SEQ ID NO:31,residues 291-398 or 629-668 of the sequence set forth in SEQ ID NO:105,or residues 473-724 or 725-968 of the sequence set forth in SEQ IDNO:107.

The term “proline-rich region” as used herein, refers to a region of aprotein kinase whose proline content over a given amino acid length ishigher than the average content of this amino acid found in proteins(i.e., >10%). Proline-rich regions are easily discernable by visualinspection of amino acid sequences and quantitated by standard computersequence analysis programs such as the DNAStar program EditSeq.Proline-rich regions have been demonstrated to participate in regulatoryprotein-protein interactions. Among these interactions, those that aremost relevant to this invention involve the “PxxP” (SEQ ID NO:148)proline rich motif found in certain protein kinases (i.e., human PAK1)and the SH3 domain of the adaptor molecule Nck (Galisteo, M. L. et al.(1996) J. Biol. Chem. 271: 20997-21000). Other regulatory interactionsinvolving “PxxP” (SEQ ID NO: 148) proline-rich motifs include the WWdomain (Sudol, M. (1996) Prog. Biochys. Mol. Bio. 65: 113-132).

The proline-rich region spans amino acid residues 527-640 of thesequence set forth in SEQ ID NO:13, residues 443-626 of the sequence setforth in SEQ ID NO:14, residues 477-680 of the sequence set forth in SEQID NO:15, residues 347-534 of the sequence set forth in SEQ ID NO:18,residues 398-628 of the sequence set forth in SEQ ID NO:105, or residues338-472 of the sequence set forth in SEQ ID NO:107.

The term “spacer region” as used herein, refers to a region of theprotein kinase located between predicted functional domains. The spacerregion has no detectable homology to any amino acid sequence in thedatabase, and can be identified by using a Smith-Waterman alignment ofthe protein sequence against the non-redundant protein database todefine the C- and N-terminal boundaries of the flanking functionaldomains. Spacer regions may or may not play a fundamental role inprotein kinase function. Precedence for the regulatory role of spacerregions in kinase function is provided by the role of the src kinasespacer in inter-domain interactions (Xu, W. et al. (1997) Nature 385:595-602).

The spacer region spans amino acid residues 641-896 of the sequence setforth in SEQ ID NO:13, residues 627-954 of the sequence set forth in SEQID NO:14, residues 681-983 of the sequence set forth in SEQ ID NO:15,residues 274-346 of the sequence set forth in SEQ ID NO:18, residues278-427 or 638-751 of the sequence set forth in SEQ ID NO:22, residues67-215 or 426-539 of the sequence set forth in SEQ ID NO:23, residues274-422 or 633-748 of the sequence set forth in SEQ ID NO:24, residues225-393 of the sequence set forth in SEQ ID NO:29, residues 282-430 or641-754 of the sequence set forth in SEQ ID NO:31, residues 174-307 ofthe sequence set forth in SEQ ID NO:103, residues 669-872 of thesequence set forth in SEQ ID NO:105, or residues 295-337 of the sequenceset forth in SEQ ID NO:107.

The term “insert” as used herein refers to a portion of a protein kinasethat is absent from a close homolog. Inserts may or may not by theproduct alternative splicing of exons. Inserts can be identified byusing a Smith-Waterman sequence alignment of the protein sequenceagainst the non-redundant protein database, or by means of a multiplesequence alignment of homologous sequences using the DNAStar programMegalign. Inserts may play a functional role by presenting a newinterface for protein-protein interactions, or by interfering with suchinteractions. Inserts span amino acid residues 52-224 of the sequenceset forth in SEQ ID NO:29 or residues 53-173 of the sequence set forthin SEQ ID NO:103.

The term “C-terminal tail” as used herein, refers to a C-terminal domainof a protein kinase, that by homology extends or protrudes past theC-terminal amino acid of its closest homolog. C-terminal tails can beidentified by using a Smith-Waterman sequence alignment of the proteinsequence against the non-redundant protein database, or by means of amultiple sequence alignment of homologous sequences using the DNAStarprogram Megalign. Depending on its length, a C-terminal tail may or maynot play a regulatory role in kinase function.

The C-terminal tail spans amino acid residues 490-516 of the sequenceset forth in SEQ ID NO:6, residues 787-887 of the sequence set forth inSEQ ID NO:23, residues 659-681 of the sequence set forth in SEQ IDNO:29, residues 994-1093 of the sequence set forth in SEQ ID NO:31, orresidues 573-591 of the sequence set forth in SEQ ID NO:103.

Various low or high stringency hybridization conditions may be useddepending upon the specificity and selectivity desired. These conditionsare well-known to those skilled in the art. Under stringenthybridization conditions only highly complementary nucleic acidsequences hybridize. Preferably, such conditions prevent hybridizationof nucleic acids having more than 1 or 2 mismatches out of 20 contiguousnucleotides, more preferably, such conditions prevent hybridization ofnucleic acids having more than 1 or 2 mismatches out of 50 contiguousnucleotides, most preferably, such conditions prevent hybridization ofnucleic acids having more than 1 or 2 mismatches out of 100 contiguousnucleotides. In some instances, the conditions may prevent hybridizationof nucleic acids having more than 5 mismatches in the full-lengthsequence.

By stringent hybridization assay conditions is meant hybridization assayconditions at least as stringent as the following: hybridization in 50%formamide, 5×SSC, 50 mM NaH₂PO₄, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicatedsalmon sperm DNA, and 5× Denhart solution at 42° C. overnight; washingwith 2×SSC, 0.1% SDS at 45 □C; and washing with 0.2×SSC, 0.1% SDS at 45°C. Under some of the most stringent hybridization assay conditions, thesecond wash can be done with 0.1×SSC at a temperature up to 70° C.(Berger et al. (1987) Guide to Molecular Cloning Techniques pg 421,hereby incorporated by reference herein including any figures, tables,or drawings.). However, other applications may require the use ofconditions falling between these sets of conditions. Methods ofdetermining the conditions required to achieve desired hybridizationsare well-known to those with ordinary skill in the art, and are based onseveral factors, including but not limited to, the sequences to behybridized and the samples to be tested.

In other preferred embodiments, the invention features isolated,enriched, or purified nucleic acid molecules encoding kinasepolypeptides, further comprising a vector or promoter effective toinitiate transcription in a host cell. The invention also featuresrecombinant nucleic acid, preferably in a cell or an organism. Therecombinant nucleic acid may contain a sequence set forth in SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27,SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104,or SEQ ID NO:106, or a functional derivative thereof and a vector or apromoter effective to initiate transcription in a host cell. Therecombinant nucleic acid can alternatively contain a transcriptionalinitiation region functional in a cell, a sequence complementary to anRNA sequence encoding a kinase polypeptide and a transcriptionaltermination region functional in a cell. Specific vectors and host cellcombinations are discussed herein.

The term “vector” relates to a single or double-stranded circularnucleic acid molecule that can be transfected into cells and replicatedwithin or independently of a cell genome. A circular double-strandednucleic acid molecule can be cut and thereby linearized upon treatmentwith restriction enzymes. An assortment of nucleic acid vectors,restriction enzymes, and the knowledge of the nucleotide sequences cutby restriction enzymes are readily available to those skilled in theart. A nucleic acid molecule encoding a kinase can be inserted into avector by cutting the vector with restriction enzymes and ligating thetwo pieces together.

The term “transfecting” defines a number of methods to insert a nucleicacid vector or other nucleic acid molecules into a cellular organism.These methods involve a variety of techniques, such as treating thecells with high concentrations of salt, an electric field, detergent, orDMSO to render the outer membrane or wall of the cells permeable tonucleic acid molecules of interest or use of various viral transductionstrategies.

The term “promoter” as used herein, refers to nucleic acid sequenceneeded for gene sequence expression. Promoter regions vary from organismto organism, but are well known to persons skilled in the art fordifferent organisms. For example, in prokaryotes, the promoter regioncontains both the promoter (which directs the initiation of RNAtranscription) as well as the DNA sequences which, when transcribed intoRNA, will signal synthesis initiation. Such regions will normallyinclude those 5′-non-coding sequences involved with initiation oftranscription and translation, such as the TATA box, capping sequence,CAAT sequence, and the like.

In preferred embodiments, the isolated nucleic acid comprises, consistsessentially of, or consists of a nucleic acid sequence set forth in SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100 SEQ ID NO:102, SEQ IDNO:104, or SEQ ID NO:106, or the corresponding full-length sequence,encodes the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQID NO:107, or the corresponding full-length amino acid sequence, afunctional derivative thereof, or at least 40, 45, 50, 60, 100, 200, or300 contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7,or of the corresponding full-length amino acid sequence; at least 250,255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, or of the corresponding full-length amino acidsequence; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous aminoacids of SEQ ID NO:18; at least 16, 25, 35, 50, 100, 200, or 300contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQID NO:29, SEQ ID NO:31, or SEQ ID NO:103, or of the correspondingfull-length amino acid sequence; 6 (preferably 10, more preferably 15,most preferably 25) or more contiguous amino acids set forth in theamino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, or thecorresponding full-length amino acid sequence; 22 (preferably 30, morepreferably 35, most preferably 45) or more contiguous amino acids setforth in the amino acid sequence of SEQ ID NO:101, or the correspondingfull-length amino acid sequence; or at least 80, 85, 90, 100, 200, or300 contiguous amino acids of SEQ ID NO:107, or functional derivativesthereof. The kinase polypeptides, selected from the group consisting ofSTLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2,SULU1, SULU3, GEK2, PAK4, and PAK5, comprise, consist essentially of, orconsist of at least at least 40, 45, 50, 60, 100, 200, or 300 contiguousamino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; at least 250,255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, or SEQ ID NO:105; at least 27, 30, 35, 40, 50, 100,200, or 300 contiguous amino acids of SEQ ID NO:18; at least 35, 40, 45,50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:103; 6(preferably 10, more preferably 15, most preferably 25) or morecontiguous amino acids set forth in the amino acid sequence of SEQ IDNO:97 or SEQ ID NO:99; 22 (preferably 30, more preferably 35, mostpreferably 45) or more contiguous amino acids set forth in the aminoacid sequence of SEQ ID NO:101; or at least 80, 85, 90, 100, 200, or 300contiguous amino acids of SEQ ID NO:107, or the correspondingfull-length sequences or derivatives thereof. The nucleic acid may beisolated from a natural source by cDNA cloning or by subtractivehybridization. The natural source may be mammalian, preferably human,blood, semen, or tissue, and the nucleic acid may be synthesized by thetriester method or by using an automated DNA synthesizer.

The term “mammal” refers preferably to such organisms as mice, rats,rabbits, guinea pigs, sheep, and goats, more preferably to cats, dogs,monkeys, and apes, and most preferably to humans.

In yet other preferred embodiments, the nucleic acid is a conserved orunique region, for example those useful for: the design of hybridizationprobes to facilitate identification and cloning of additionalpolypeptides, the design of PCR probes to facilitate cloning ofadditional polypeptides, obtaining antibodies to polypeptide regions,and designing antisense oligonucleotides.

By “conserved nucleic acid regions”, are meant regions present on two ormore nucleic acids encoding a kinase polypeptide, to which a particularnucleic acid sequence can hybridize under lower stringency conditions.Examples of lower stringency conditions suitable for screening fornucleic acid encoding kinase polypeptides are provided in Abe, et al.(J. Biol. Chem. 19: 13361-13368, 1992), hereby incorporated by referenceherein in its entirety, including any drawings, figures, or tables.Preferably, conserved regions differ by no more than 5 out of 20nucleotides, even more preferably 2 out of 20 nucleotides or mostpreferably 1 out of 20 nucleotides.

By “unique nucleic acid region” is meant a sequence present in a nucleicacid coding for a kinase polypeptide that is not present in a sequencecoding for any other naturally occurring polypeptide. Such regionspreferably encode 32 (preferably 40, more preferably 45, most preferably55) or more contiguous amino acids set forth in the amino acid sequenceof SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or the correspondingfull-length amino acid sequence; 250 (preferably 255, more preferably260, most preferably 270) or more contiguous amino acids set forth inthe amino acid sequence SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, orSEQ ID NO:105, or the corresponding full-length amino acid sequence; 27(preferably 30, more preferably 40, most preferably 45) or morecontiguous amino acids set forth in the amino acid sequence SEQ IDNO:18; 16 (preferably 20, more preferably 25, most preferably 35) ormore contiguous amino acids set forth in the amino acid sequence SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, or SEQ IDNO:103, or the corresponding full-length amino acid sequence; 6(preferably 10, more preferably 15, most preferably 25) or morecontiguous amino acids set forth in the amino acid sequence of SEQ IDNO:97 or SEQ ID NO:99, 22 (preferably 30, more preferably 35, mostpreferably 45) or more contiguous amino acids set forth in the aminoacid sequence of SEQ ID NO:101, or the corresponding full-length aminoacid sequence; or 78 (preferably 80, more preferably 85, most preferably90) or more contiguous amino acids set forth in the amino acid sequenceSEQ ID NO:107, or functional derivatives thereof. In particular, aunique nucleic acid region is preferably of mammalian origin.

A second aspect of the invention features a nucleic acid probe for thedetection of nucleic acid encoding a kinase polypeptide in a sample,wherein said polypeptide is selected from the group consisting of STLK2,STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1,SULU3, GEK2, PAK4, and PAK5. Preferably, the nucleic acid probe encodesa kinase polypeptide that is a fragment of the protein encoded by theamino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97,SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ IDNO:107, or the corresponding full-length amino acid sequences, not toinclude fragments consisting only of amino acids 1-22 of SEQ ID NO:13 oramino acids 1-33 of SEQ ID NO:107. The nucleic acid probe contains anucleotide base sequence that will hybridize to a sequence set forth inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ IDNO:104, or SEQ ID NO:106, or the corresponding full-length sequence, ora functional derivative thereof.

In preferred embodiments, the nucleic acid probe hybridizes to nucleicacid encoding at least 6, 12, 75, 90, 105, 120, 150, 200, 250, 300 or350 contiguous amino acids of the sequence set forth in SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ IDNO:31 SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, or SEQ ID NO:107, or the corresponding full-length amino acidsequence, or functional derivatives thereof.

Methods for using the probes include detecting the presence or amount ofkinase RNA in a sample by contacting the sample with a nucleic acidprobe under conditions such that hybridization occurs and detecting thepresence or amount of the probe bound to kinase RNA. The nucleic acidduplex formed between the probe and a nucleic acid sequence coding for akinase polypeptide may be used in the identification of the sequence ofthe nucleic acid detected (Nelson et al., in Nonisotopic DNA ProbeTechniques, Academic Press, San Diego, Kricka, ed., p. 275, 1992, herebyincorporated by reference herein in its entirety, including anydrawings, figures, or tables). Kits for performing such methods may beconstructed to include a container means having disposed therein anucleic acid probe.

In a third aspect, the invention describes a recombinant cell or tissuecomprising a nucleic acid molecule encoding a kinase polypeptideselected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6,STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. Insuch cells, the nucleic acid may be under the control of the genomicregulatory elements, or may be under the control of exogenous regulatoryelements including an exogenous promoter. By “exogenous” it is meant apromoter that is not normally coupled in vivo transcriptionally to thecoding sequence for the kinase polypeptides.

The polypeptide is preferably a fragment of the protein encoded by theamino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97,SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ IDNO:107, or the corresponding full-length amino acid sequence, not toinclude fragments consisting only of amino acids 1-22 of SEQ ID NO:13 oramino acids 1-33 of SEQ ID NO:107. By “fragment,” is meant an amino acidsequence present in a kinase polypeptide. Preferably, such a sequencecomprises at least 32, 45, 50, 60, 100, 200, or 300 contiguous aminoacids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or of thecorresponding full-length amino acid sequence; at least 250, 255, 275,300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, OR SEQ ID NO:105, or of the corresponding full-length amino acidsequence; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous aminoacids of SEQ ID NO:18; at least 16, 25, 35, 50, 100, 200, or 300contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQID NO:29, SEQ ID NO:31 or SEQ ID NO:103, or of the correspondingfull-length amino acid sequence; 6 (preferably 10, more preferably 15,most preferably 25) or more contiguous amino acids set forth in theamino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, 22 (preferably 30,more preferably 35, most preferably 45) or more contiguous amino acidsset forth in the amino acid sequence of SEQ ID NO:101; at least 78, 85,90, 100, 200, or 300 contiguous amino acids of SEQ ID NO:107, or thecorresponding full-length amino acid sequence; or a functionalderivative thereof.

In a fourth aspect, the invention features an isolated, enriched, orpurified kinase polypeptide selected from the group consisting of STLK2,STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4; KHS2, SULU1,SULU3, GEK2, PAK4, and PAK5.

By “isolated” in reference to a polypeptide is meant a polymer of aminoacids (2 or more amino acids) conjugated to each other, includingpolypeptides that are isolated from a natural source or that aresynthesized. The isolated polypeptides of the present invention areunique in the sense that they are not found in a pure or separated statein nature. Use of the term “isolated” indicates that a naturallyoccurring sequence has been removed from its normal cellularenvironment. Thus, the sequence may be in a cell-free solution or placedin a different cellular environment. The term does not imply that thesequence is the only amino acid chain present, but that it isessentially free (about 90-95% pure at least) of non-amino acid materialnaturally associated with it.

By the use of the term “enriched” in reference to a polypeptide is meantthat the specific amino acid sequence constitutes a significantly higherfraction (2-5 fold) of the total amino acid sequences present in thecells or solution of interest than in normal or diseased cells or in thecells from which the sequence was taken. This could be caused by aperson by preferential reduction in the amount of other amino acidsequences present, or by a preferential increase in the amount of thespecific amino acid sequence of interest, or by a combination of thetwo. However, it should be noted that enriched does not imply that thereare no other amino acid sequences present, just that the relative amountof the sequence of interest has been significantly increased. The termsignificant here is used to indicate that the level of increase isuseful to the person making such an increase, and generally means anincrease relative to other amino acid sequences of about at least2-fold, more preferably at least 5- to 10-fold or even more. The termalso does not imply that there is no amino acid sequence from othersources. The other source of amino acid sequences may, for example,comprise amino acid sequence encoded by a yeast or bacterial genome, ora cloning vector such as pUC19. The term is meant to cover only thosesituations in which man has intervened to increase the proportion of thedesired amino acid sequence.

It is also advantageous for some purposes that an amino acid sequence bein purified form. The term “purified” in reference to a polypeptide doesnot require absolute purity (such as a homogeneous preparation);instead, it represents an indication that the sequence is relativelypurer than in the natural environment. Compared to the natural levelthis level should be at least 2-5 fold greater (e.g., in terms ofmg/mL). Purification of at least one order of magnitude, preferably twoor three orders, and more preferably four or five orders of magnitude isexpressly contemplated. The substance is preferably free ofcontamination at a functionally significant level, for example 90%, 95%,or 99% pure.

In preferred embodiments, the kinase polypeptide is a fragment of theprotein encoded by the amino acid sequence set forth in SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, or SEQ ID NO:107, or the corresponding full-length amino acidsequences, not to include fragments consisting only of amino acids 1-22of SEQ ID NO:13 or amino acids 1-33 of SEQ ID NO:107. Preferably, thekinase polypeptide contains at least 32, 45, 50, 60, 100, 200, or 300contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, orthe corresponding full-length amino acid sequence; at least 250, 255,275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, or SEQ ID NO:105, or the corresponding full-length aminoacid sequence; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguousamino acids of SEQ ID NO:18; at least 16, 25, 35, 50, 100, 200, or 300contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQID NO:29, SEQ ID NO:31, or SEQ ID NO:103, or the correspondingfull-length amino acid sequence; 6 (preferably 10, more preferably 15,most preferably 25) or more contiguous amino acids set forth in theamino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, 22 (preferably 30,more preferably 35, most preferably 45) or more contiguous amino acidsset forth in the amino acid sequence of SEQ ID NO:101, or thecorresponding full-length amino acid sequence; or at least 78, 85, 90,100, 200, or 300 contiguous amino acids of SEQ ID NO:107, or afunctional derivative thereof.

In preferred embodiments, the kinase polypeptide comprises an amino acidsequence having (a) the amino acid sequence set forth in SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, or SEQ ID NO:107; (b) the amino acid sequence set forth in SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:103, SEQ IDNO:105, or SEQ ID NO:107, except that it lacks one or more, but not all,of the following segments of amino acid residues: 1-21, 22-274, or275-416 of SEQ ID NO:5, 1-31, 32-308, 309-489 or 490-516 of SEQ ID NO:6,1-178 or 179-414 of SEQ ID NO:7, 1-22, 23-289, 290-526, 527-640,641-896, or 897-1239 of SEQ ID NO:13, 1-255, 256-442, 443-626, 627-954,or 955-1297 of SEQ ID NO:14, 1-255, 256-476, 477-680, 681-983, or984-1326 of SEQ ID NO:15, 1-13, 14-273, 274-346, 347-534, or 535-894 ofSEQ ID NO:18, 1-21, 22-277, 278-427, 428-637, 638-751, or 752-898 of SEQID NO:22, 1-66, 67-215, 216-425, 426-539, 540-786, or 787-887 of SEQ IDNO:23, 1-25, 26-273, 274-422, 423-632, or 633-748 of SEQ ID NO:24, 1-51,52-224, 225-393, 394-658, or 659-681 of SEQ ID NO:29, 1-25, 26-281,282-430, 431-640, 641-754, 755-901, or 902-1001 of SEQ ID NO:31, 1-10,11-321, or 322-373 of SEQ ID NO:97, 1-57, 58-369, or 370-418 of SEQ IDNO:99, 1-52, 53-173,174-307, 308-572, or 573-591 of SEQ ID NO:103, 1-24,25-289, 290-397, 398-628, 629-668, 669-872, or 873-1227 of SEQ IDNO:105, or 1-33, 34-294, 295-337, 338-472, 473-724, or 725-968 of SEQ IDNO:107; (c) the amino acid sequence set forth in SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107 fromamino acid residues 1-21, 22-274, or 275-416 of SEQ ID NO:5, 1-31,32-308, 309-489, or 490-516 of SEQ ID NO:6, 1-178 or 179-414 of SEQ IDNO:7, 23-289, 290-526, 527-640, 641-896, or 897-1239 of SEQ ID NO:13,1-255, 256-442, 443-626, 627-954, or 955-1297 of SEQ ID NO:14, 1-255,256-476, 477-680, 681-983, or 984-1326 of SEQ ID NO:15, 1-13, 14-273,274-346, 347-534, or 535-894 of SEQ ID NO:18, 1-21, 22-277, 278-427,428-637, 638-751, or 752-898 of SEQ ID NO:22, 1-66, 67-215, 216-425,426-539, 540-786, or 787-887 of SEQ ID NO:23, 1-25, 26-273, 274-422,423-632, or 633-748 of SEQ ID NO:24, 1-51, 52-224, 225-393, 394-658, or659-681 of SEQ ID NO:29, 1-25, 26-273, 274-422, 423-632, 633-746,747-993, or 994-1093 of SEQ ID NO:31, 1-10, 11-321, or 322-373 of SEQ IDNO:97, 1-57, 58-369, or 370-418 of SEQ ID NO:99, 1-52, 53-173, 174-307,308-572, or 573-591 of SEQ ID NO:103, 1-24, 25-289, 290-397, 398-628,629-668, 669-872, or 873-1227 of SEQ ID NO:105, or 1-33, 34-294,295-337, 338-472, 473-724, or 725-968 of SEQ ID NO:107; or (d) the aminoacid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107,except that it lacks one or more, but not all, of the domains selectedfrom the group consisting of a C-terminal domain, a catalytic domain, anN-terminal domain, a spacer region, a proline-rich region, a coiled-coilstructure region, an insert, and a C-terminal tail.

The polypeptide can be isolated from a natural source by methodswell-known in the art. The natural source may be mammalian, preferablyhuman, blood, semen, or tissue, and the polypeptide may be synthesizedusing an automated polypeptide synthesizer. The isolated, enriched, orpurified kinase polypeptide is preferably: a STLK2, STLK3, STLK4, STLK5,STLK6, or STLK7 polypeptide; a ZC1, ZC2, ZC3, or ZC4 polypeptide; a KHS2polypeptide; a SULU1 or SULU3 polypeptide; a GEK2 polypeptide; or a PAK4or PAK5 polypeptide.

In some embodiments the invention includes a recombinant kinasepolypeptide selected from the group consisting of STLK2, STLK3, STLK4,STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4,and PAK5. By “recombinant kinase polypeptide” is meant a polypeptideproduced by recombinant DNA techniques such that it is distinct from anaturally occurring polypeptide either in its location (e.g., present ina different cell or tissue than found in nature), purity or structure.Generally, such a recombinant polypeptide will be present in a cell inan amount different from that normally observed in nature.

In a fifth aspect, the invention features an antibody (e.g., amonoclonal or polyclonal antibody) having specific binding affinity to akinase polypeptide or a kinase polypeptide domain or fragment where thepolypeptide is selected from the group consisting of STLK2, STLK3,STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3,GEK2, PAK4, and PAK5. By “specific binding affinity” is meant that theantibody binds to the target kinase polypeptide with greater affinitythan it binds to other polypeptides under specified conditions.Antibodies or antibody fragments are polypeptides that contain regionsthat can bind other polypeptides. The term “specific binding affinity”describes an antibody that binds to a kinase polypeptide with greateraffinity than it binds to other polypeptides under specified conditions.

The term “polyclonal” refers to antibodies that are heterogenouspopulations of antibody molecules derived from the sera of animalsimmunized with an antigen or an antigenic functional derivative thereof.For the production of polyclonal antibodies, various host animals may beimmunized by injection with the antigen. Various adjuvants may be usedto increase the immunological response, depending on the host species.

“Monoclonal antibodies” are substantially homogenous populations ofantibodies to a particular antigen. They may be obtained by anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. Monoclonal antibodies may be obtainedby methods known to those skilled in the art (Kohler et al., Nature 256:495-497, 1975, and U.S. Pat. No. 4,376,110, both of which are herebyincorporated by reference herein in their entirety including anyfigures, tables, or drawings).

The term “antibody fragment” refers to a portion of an antibody, oftenthe hyper variable region and portions of the surrounding heavy andlight chains, that displays specific binding affinity for a particularmolecule. A hyper variable region is a portion of an antibody thatphysically binds to the polypeptide target.

Antibodies or antibody fragments having specific binding affinity to akinase polypeptide of the invention may be used in methods for detectingthe presence and/or amount of kinase polypeptide in a sample by probingthe sample with the antibody under conditions suitable forkinase-antibody immunocomplex formation and detecting the presenceand/or amount of the antibody conjugated to the kinase polypeptide.Diagnostic kits for performing such methods may be constructed toinclude antibodies or antibody fragments specific for the kinase as wellas a conjugate of a binding partner of the antibodies or the antibodiesthemselves.

An antibody or antibody fragment with specific binding affinity to akinase polypeptide of the invention can be isolated, enriched, orpurified from a prokaryotic or eukaryotic organism. Routine methodsknown to those skilled in the art enable production of antibodies orantibody fragments, in both prokaryotic and eukaryotic organisms.Purification, enrichment, and isolation of antibodies, which arepolypeptide molecules, are described above.

Antibodies having specific binding affinity to a kinase polypeptide ofthe invention may be used in methods for detecting the presence and/oramount of kinase polypeptide in a sample by contacting the sample withthe antibody under conditions such that an immunocomplex forms anddetecting the presence and/or amount of the antibody conjugated to thekinase polypeptide. Diagnostic kits for performing such methods may beconstructed to include a first container containing the antibody and asecond container having a conjugate of a binding partner of the antibodyand a label, such as, for example, a radioisotope. The diagnostic kitmay also include notification of an FDA approved use and instructionstherefor.

In a sixth aspect, the invention features a hybridoma which produces anantibody having specific binding affinity to a kinase polypeptide or akinase polypeptide domain, where the polypeptide is selected from thegroup consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2,ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. By “hybridoma” ismeant an immortalized cell line that is capable of secreting anantibody, for example an antibody to a kinase of the invention. Inpreferred embodiments, the antibody to the kinase comprises a sequenceof amino acids that is able to specifically bind a kinase polypeptide ofthe invention.

In a seventh aspect, the invention features a kinase polypeptide bindingagent able to bind to a kinase polypeptide selected from the groupconsisting of STLK2, STLK3, STLK4, STLK6, STLK7, STLK5, ZC1, ZC2, ZC3,ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. The binding agent ispreferably a purified antibody that recognizes an epitope present on akinase polypeptide of the invention. Other binding agents includemolecules that bind to kinase polypeptides and analogous molecules thatbind to a kinase polypeptide. Such binding agents may be identified byusing assays that measure kinase binding partner activity, such as thosethat measure PDGFR activity.

The invention also features a method for screening for human cellscontaining a kinase polypeptide of the invention or an equivalentsequence. The method involves identifying the novel polypeptide in humancells using techniques that are routine and standard in the art, such asthose described herein for identifying the kinases of the invention(e.g., cloning, Southern or Northern blot analysis, in situhybridization, PCR amplification, etc.).

In an eighth aspect, the invention features methods for identifying asubstance that modulates kinase activity comprising the steps of: (a)contacting a kinase polypeptide selected from the group consisting ofSTLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2,SULU1, SULU3, GEK2, PAK4, and PAK5 with a test substance; (b) measuringthe activity of said polypeptide; and (c) determining whether saidsubstance modulates the activity of said polypeptide.

The term “modulates” refers to the ability of a compound to alter thefunction of a kinase of the invention. A modulator preferably activatesor inhibits the activity of a kinase of the invention depending on theconcentration of the compound exposed to the kinase.

The term “activates” refers to increasing the cellular activity of thekinase. The term inhibit refers to decreasing the cellular activity ofthe kinase. Kinase activity is preferably the interaction with a naturalbinding partner.

The term “modulates” also refers to altering the function of kinases ofthe invention by increasing or decreasing the probability that a complexforms between the kinase and a natural binding partner. A modulatorpreferably increases the probability that such a complex forms betweenthe kinase and the natural binding partner, more preferably increases ordecreases the probability that a complex forms between the kinase andthe natural binding partner depending on the concentration of thecompound exposed to the kinase, and most preferably decreases theprobability that a complex forms between the kinase and the naturalbinding partner.

The term “complex” refers to an assembly of at least two molecules boundto one another. Signal transduction complexes often contain at least twoprotein molecules bound to one another. For instance, a protein tyrosinereceptor protein kinase, GRB2, SOS, RAF, and RAS assemble to form asignal transduction complex in response to a mitogenic ligand.

The term “natural binding partner” refers to polypeptides, lipids, smallmolecules, or nucleic acids that bind to kinases in cells. A change inthe interaction between a kinase and a natural binding partner canmanifest itself as an increased or decreased probability that theinteraction forms, or an increased or decreased concentration ofkinase/natural binding partner complex.

The term “contacting” as used herein refers to mixing a solutioncomprising the test compound with a liquid medium bathing the cells ofthe methods. The solution comprising the compound may also compriseanother component, such as dimethyl sulfoxide (DMSO), which facilitatesthe uptake of the test compound or compounds into the cells of themethods. The solution comprising the test compound may be added to themedium bathing the cells by utilizing a delivery apparatus, such as apipet-based device or syringe-based device.

In a ninth aspect, the invention features methods for identifying asubstance that modulates kinase activity in a cell comprising the stepsof: (a) expressing a kinase polypeptide in a cell, wherein saidpolypeptide is selected from the group consisting of STLK2, STLK3,STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3,GEK2, PAK4, and PAK5; (b) adding a test substance to said cell; and (c)monitoring a change in cell phenotype or the interaction between saidpolypeptide and a natural binding partner.

The term “expressing” as used herein refers to the production of kinasesof the invention from a nucleic acid vector containing kinase geneswithin a cell. The nucleic acid vector is transfected into cells usingwell known techniques in the art as described herein.

In a tenth aspect, the invention provides methods for treating a diseaseby administering to a patient in need of such treatment a substance thatmodulates the activity of a kinase selected from the group consisting ofSTLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2,SULU1, SULU3, GEK2, PAK4, and PAK5. Preferably, the disease is selectedfrom the group consisting of immune-related diseases and disorders,organ transplantation, myocardial infarction, cardiovascular disease,stroke, renal failure, oxidative stress-related neurodegenerativedisorders, and cancer. Most preferably, the immune-related diseases anddisorders include, but are not limited to, rheumatoid arthritis,artherosclerosis, and autoimmune disorders.

In preferred embodiments, the invention provides methods for treating orpreventing a disease or disorder by administering to a patient in needof such treatment a substance that modulates the activity of a kinasepolypeptide selected from the group consisting of ZC1, ZC2, ZC3, ZC4,KHS2, PAK4, and PAK5. Preferably, the disease or disorder is selectedfrom the group consisting of rheumatoid arthritis, artherosclerosis,autoimmune disorders, and organ transplantation. The invention alsofeatures methods of treating or preventing a disease or disorder byadministering to a patient in need of such treatment a substance thatmodulates the activity of a kinase polypeptide selected from the groupconsisting of STLK1, STLK2, STLK3, STLK4, STLK5, STLK6, and STLK7.Preferably the disease or disorder is selected from the group consistingof immune-related diseases and disorders, myocardial infarction,cardiomyopathies, stroke, renal failure, and oxidative stress-relatedneurodegenerative disorders. Most preferably, the immune-relateddiseases and disorders are selected from the group consisting ofrheumatoid arthritis, chronic inflammatory bowel disease, chronicinflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis,psoriasis, atherosclerosis, rhinitis, autoimmunity, and organtransplantation.

The invention also features methods of treating or preventing a diseaseor disorder by administering to a patient in need of such treatment asubstance that modulates the activity of a kinase polypeptide selectedfrom the group consisting of ZC1, ZC2, ZC3, and ZC4. Preferably thedisease is selected from the group consisting of immune-related diseasesand disorders, cardiovascular disease, and cancer. Most preferably, theimmune-related diseases and disorders are selected from the groupconsisting of rheumatoid arthritis, chronic inflammatory bowel disease,chronic inflammatory pelvic disease, multiple sclerosis, asthma,osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, andorgan transplantation.

Substances useful for treatment of kinase-related disorders or diseasespreferably show positive results in one or more in vitro assays for anactivity corresponding to treatment of the disease or disorder inquestion (Examples of such assays are provided in the references insection VI, below; and in Example 7, herein). Examples of substancesthat can be screened for favorable activity are provided and referencedin section VI, below. The substances that modulate the activity of thekinases preferably include, but are not limited to, antisenseoligonucleotides and inhibitors of protein kinases, as determined bymethods and screens referenced in section VI and Example 7, below.

The term “preventing” refers to decreasing the probability that anorganism contracts or develops an abnormal condition.

The term “treating” refers to having a therapeutic effect and at leastpartially alleviating or abrogating an abnormal condition in theorganism.

The term “therapeutic effect” refers to the inhibition or activationfactors causing or contributing to the abnormal condition. A therapeuticeffect relieves to some extent one or more of the symptoms of theabnormal condition. In reference to the treatment of abnormalconditions, a therapeutic effect can refer to one or more of thefollowing: (a) an increase in the proliferation, growth, and/ordifferentiation of cells; (b) inhibition (i.e., slowing or stopping) ofcell death; (c) inhibition of degeneration; (d) relieving to some extentone or more of the symptoms associated with the abnormal condition; and(e) enhancing the function of the affected population of cells.Compounds demonstrating efficacy against abnormal conditions can beidentified as described herein.

The term “abnormal condition” refers to a function in the cells ortissues of an organism that deviates from their normal functions in thatorganism. An abnormal condition can relate to cell proliferation, celldifferentiation, or cell survival.

Abnormal cell proliferative conditions include cancers such as fibroticand mesangial disorders, abnormal angiogenesis and vasculogenesis, woundhealing, psoriasis, diabetes mellitus, and inflammation.

Abnormal differentiation conditions include, but are not limited toneurodegenerative disorders, slow wound healing rates, and slow tissuegrafting healing rates.

Abnormal cell survival conditions relate to conditions in whichprogrammed cell death (apoptosis) pathways are activated or abrogated. Anumber of protein kinases are associated with the apoptosis pathways.Aberrations in the function of any one of the protein kinases could leadto cell immortality or premature cell death.

The term “aberration”, in conjunction with the function of a kinase in asignal transduction process, refers to a kinase that is over- orunder-expressed in an organism, mutated such that its catalytic activityis lower or higher than wild-type protein kinase activity, mutated suchthat it can no longer interact with a natural binding partner, is nolonger modified by another protein kinase or protein phosphatase, or nolonger interacts with a natural binding partner.

The term “administering” relates to a method of incorporating a compoundinto cells or tissues of an organism. The abnormal condition can beprevented or treated when the cells or tissues of the organism existwithin the organism or outside of the organism. Cells existing outsidethe organism can be maintained or grown in cell culture dishes. Forcells harbored within the organism, many techniques exist in the art toadminister compounds, including (but not limited to) oral, parenteral,dermal, injection, and aerosol applications. For cells outside of theorganism, multiple techniques exist in the art to administer thecompounds, including (but not limited to) cell microinjectiontechniques, transformation techniques, and carrier techniques.

The abnormal condition can also be prevented or treated by administeringa compound to a group of cells having an aberration in a signaltransduction pathway to an organism. The effect of administering acompound on organism function can then be monitored. The organism ispreferably a mouse, rat, rabbit, guinea pig, or goat, more preferably amonkey or ape, and most preferably a human.

In an eleventh aspect, the invention features methods for detection of akinase polypeptide in a sample as a diagnostic tool for diseases ordisorders, wherein the method comprises the steps of: (a) contacting thesample with a nucleic acid probe which hybridizes under hybridizationassay conditions to a nucleic acid target region of a kinase polypeptideselected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6,STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5,said probe comprising the nucleic acid sequence encoding thepolypeptide, fragments thereof, and the complements of the sequences andfragments; and (b) detecting the presence or amount of the probe:targetregion hybrid as an indication of the disease.

In preferred embodiments of the invention, the disease or disorder isselected from the group consisting of rheumatoid arthritis,artherosclerosis, autoimmune disorders, organ transplantation,myocardial infarction, cardiomyopathies, stroke, renal failure,oxidative stress-related neurodegenerative disorders, and cancer. Inother preferred embodiments, the kinase polypeptide is selected from thegroup consisting of PAK4 and PAK5, or the polypeptide is selected fromthe group consisting of ZC1, ZC2, ZC3, and ZC4, and the disease iscancer.

The kinase “target region” is the nucleotide base sequence set forth inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO: 10, SEQID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ IDNO:104, or SEQ ID NO:106, or the corresponding full-length sequences, afunctional derivative thereof, or a fragment thereof to which thenucleic acid probe will specifically hybridize. Specific hybridizationindicates that in the presence of other nucleic acids the probe onlyhybridizes detectably with the kinase of the invention's target region.Putative target regions can be identified by methods well known in theart consisting of alignment and comparison of the most closely relatedsequences in the database.

In preferred embodiments the nucleic acid probe hybridizes to a kinasetarget region encoding at least 6, 12, 75, 90, 105, 120, 150, 200, 250,300 or 350 contiguous amino acids of the sequence set forth in SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ IDNO:103, SEQ ID NO:105, or SEQ ID NO:107, or the correspondingfull-length amino acid sequence, or a functional derivative thereof.Hybridization conditions should be such that hybridization occurs onlywith the kinase genes in the presence of other nucleic acid molecules.Under stringent hybridization conditions only highly complementarynucleic acid sequences hybridize. Preferably, such conditions preventhybridization of nucleic acids having more than 1 or 2 mismatches out of20 contiguous nucleotides. Such conditions are defined supra.

The diseases for which detection of kinase genes in a sample could bediagnostic include diseases in which kinase nucleic acid (DNA and/orRNA) is amplified in comparison to normal cells. By “amplification” ismeant increased numbers of kinase DNA or RNA in a cell compared withnormal cells. In normal cells, kinases are typically found as singlecopy genes. In selected diseases, the chromosomal location of the kinasegenes may be amplified, resulting in multiple copies of the gene, oramplification. Gene amplification can lead to amplification of kinaseRNA, or kinase RNA can be amplified in the absence of kinase DNAamplification.

“Amplification” as it refers to RNA can be the detectable presence ofkinase RNA in cells, since in some normal cells there is no basalexpression of kinase RNA. In other normal cells, a basal level ofexpression of kinase exists, therefore in these cases amplification isthe detection of at least 1-2-fold, and preferably more, kinase RNA,compared to the basal level.

The diseases that could be diagnosed by detection of kinase nucleic acidin a sample preferably include cancers. The test samples suitable fornucleic acid probing methods of the present invention include, forexample, cells or nucleic acid extracts of cells, or biological fluids.The samples used in the above-described methods will vary based on theassay format, the detection method and the nature of the tissues, cellsor extracts to be assayed. Methods for preparing nucleic acid extractsof cells are well known in the art and can be readily adapted in orderto obtain a sample that is compatible with the method utilized.

In a final aspect, the invention features a method for detection of akinase polypeptide in a sample as a diagnostic tool for a disease ordisorder, wherein the method comprises: (a) comparing a nucleic acidtarget region encoding the kinase polypeptide in a sample, where thekinase polypeptide is selected from the group consisting of STLK2,STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1,SULU3, GEK2, PAK4, and PAK5, or one or more fragments thereof, with acontrol nucleic acid target region encoding the kinase polypeptide, orone or more fragments thereof; and (b) detecting differences in sequenceor amount between the target region and the control target region, as anindication of the disease or disorder. Preferably, the disease ordisorder is selected from the group consisting of immune-relateddiseases and disorders, organ transplantation, myocardial infarction,cardiovascular disease, stroke, renal failure, oxidative stress-relatedneurodegenerative disorders, and cancer. Immune-related diseases anddisorders include, but are not limited to, those discussed previously.

The term “comparing” as used herein refers to identifying discrepanciesbetween the nucleic acid target region isolated from a sample, and thecontrol nucleic acid target region. The discrepancies can be in thenucleotide sequences, e.g. insertions, deletions, or point mutations, orin the amount of a given nucleotide sequence. Methods to determine thesediscrepancies in sequences are well-known to one of ordinary skill inthe art. The “control” nucleic acid target region refers to the sequenceor amount of the sequence found in normal cells, e.g. cells that are notdiseased as discussed previously.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. For example, insome instances the nucleotide sequence of the ZC4 kinase polypeptide maynot be part of a preferred embodiment.

The summary of the invention described above is not limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B and 1C show a multiple sequence alignment of the amino acidsequences (SEQ ID NOS 84-85, 5-7, respectively, in order of appearance)of the STE20-STE20 family kinases.

FIGS. 2A and 2B show a multiple sequence alignment of the amino acidsequences (SEQ ID NOS 84, 86-87 & 8, respectively, in order ofappearance) of the STE20-STLK5 family kinases.

FIGS. 3A, 3B, 3C, 3D, 3E, 3F and 3G show a multiple sequence alignmentof the amino acid sequences (SEQ ID NOS 88-89, 13-16, respectively, inorder of appearance) of STE20-ZC family kinases.

FIGS. 4A, 4B and 4C show a pairwise sequence (SEQ ID NOS 91 & 18,respectively, in order of appearance) alignment of STE20-KHS familykinases.

FIGS. 5A, 5B, 5C and 5D show a multiple sequence alignment of the aminoacid sequences (SEQ ID NOS 90, 22, 24 & 151 respectively, in order ofappearance) of STE20-SULU family kinases.

FIGS. 6A, 6B and 6C show a pairwise sequence (SEQ ID NOS 92 & 26,respectively, in order of appearance) alignment of STE20-GEK familykinases.

FIGS. 7A, 7B and 7C show a multiple sequence alignment of the amino acidsequences (SEQ ID NOS 93-95, 29-30 respectively, in order of appearance)of STE20-PAK family kinases.

FIGS. 8A, 8B, 8C, 8D, 8E, 8F and 8G show the amino acid sequences ofhuman STLK2(SEQ ID NO:5), human STLK3(SEQ ID NO:6), human STLK4(SEQ IDNO:7), human STLK5(SEQ ID NO:8), human ZC1(SEQ ID NO:13), human ZC2(SEQID NO:14), human ZC3(SEQ ID NO:15), human ZC4(SEQ ID NO:16), humanKHS2(SEQ ID NO:18), human SULU1(SEQ ID NO:22), human SULU3(SEQ IDNO:23), murine SULU3(SEQ ID NO:24), human GEK2(SEQ ID NO:26), humanPAK4(SEQ ID NO:29), and human PAK5(SEQ ID NO30).

FIGS. 9A, 9B, 9C, 9D, 9E, 9F, 9G, 9H, 9I, 9J, 9K, 9L, 9M, 9N, 9O, 9P,9Q, 9R, 9S, 9T, 9U and 9V show the nucleic acid sequences of humanSTLK2(SEQ ID NO:1), human STLK3(SEQ ID NO:2), human STLK4(SEQ ID NO:3),human STLK5(SEQ ID NO:4), human ZC1(SEQ ID NO:9), human ZC2(SEQ IDNO:10), human ZC3(SEQ ID NO:11), human ZC4(SEQ ID NO:12), human KHS2(SEQID NO:17), human SULU1(SEQ ID NO:19), human SULU3(SEQ ID NO:20), murineSULU3(SEQ ID NO:21), human GEK2(SEQ ID NO:25), human PAK4(SEQ ID NO:27),and human PAK5(SEQ ID NO:28).

FIGS. 10A, 10B and 10C show the full-length amino acid sequences ofhuman STLK5 (SEQ ID NO: 97), human PAK5 (SEQ ID NO:103), and human ZC4(SEQ ID NO:105), as well as the partial amino acid sequences of humanfull-length STLK6 (SEQ ID NO: 99) and human STLK7 (SEQ ID NO: 101) andhuman GEK2 (SEQ ID NO: 107).

FIGS. 11A, 11B, 11C, 11D, 11E, 11F, 11G and 11H show the full-lengthnucleic acid sequences of human STLK5 (SEQ ID NO:96), human PAK5 (SEQ IDNO:102), and human ZC4 (SEQ ID NO:104), as well as the partial nucleicacid sequences of human STLK6 (SEQ ID NO: 98) and human STLK7 (SEQ IDNO: 100) and human GEK2 (SEQ ID NO: 106).

FIGS. 12A and 12B show a multiple sequence alignment among human SPAK(SEQ ID NO: 153), human STLK6 (SEQ ID NO: 99), human STLK7 (SEQ ID NO:101) and full-length human STLK5 (SEQ ID NO: 152).

FIGS. 13A, 13B and 13C show a multiple sequence alignment among humanPAK1 (SEQ ID NO: 93), human PAK4 (SEQ ID NO: 29) and human PAK5 (SEQ IDNO: 103).

FIGS. 14A, 14B and 14C show a pair-wise sequence alignment between humanZC1 (SEQ ID NO: 15) and human ZC4 (SEQ ID NO: 105).

FIGS. 15A, 15B and 15C show a pair-wise sequence alignment between LOK1(SEQ ID NO: 154) and full-length GEK2 (SEQ ID NO: 155).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in part to kinase polypeptides, nucleicacids encoding such polypeptides, cells containing such nucleic acids,antibodies to such polypeptides, assays utilizing such polypeptides, andmethods relating to all of the foregoing. The present invention is basedupon the isolation and characterization of new kinase polypeptides. Thepolypeptides and nucleic acids may be produced using well-known andstandard synthesis techniques when given the sequences presented herein.

The recent elucidation of the DNA sequence of Saccharomyces cerevesiaehas provided the first complete example of the genetic informationcontained in a simple eukaryotic organism. Analysis of this yeast genomerevealed that it contains at least 113 protein kinases. These kinaseswere further subdivided into several structurally related groups. One ofthese newly defined groups was termed the STE20-family to represent itsfounding member STE20, which is a protein kinase involved in the yeastpheromone response pathway that initiates a protein kinase cascade inresponse to a G-protein mediated signal. S. cerevesiae has twoadditional members of this family, CLA4, and YOL113W (HRA655).

Several mammalian homologues have recently been identified that belongto the STE20-family, including SOK-1 (human STE20), GC-kinase, KHS,HPK1, NIK, SLK, GEK, PAK1, PAK65, MST1, and CDC7. Furthermore, theDrosophila and the C. elegans genome efforts have identified additionalprotein kinases which belong to the STE20-family, yet have structurallyunique extracatalytic domains, including ZC504.4 and SULU kinases fromC. elegans, and NINAC of Drosophila.

STE20-related protein kinases have been implicated as regulating avariety of cellular responses, including response to growth factors orcytokines, oxidative-, UV-, or irradiation-related stress pathways,inflammatory signals (i.e., TNF□), apoptotic stimuli (i.e., Fas), T andB cell costimulation, the control of cytoskeletal architecture, andcellular transformation. Typically, the STE20-related kinases serve asupstream regulators of MAPK cascades. Examples include: HPK1, aprotein-serine/threonine kinase (STK) that possesses a STE20-like kinasedomain that activates a protein kinase pathway leading to thestress-activated protein kinase SAPK/JNK; PAK1, an STK with an upstreamCDC42-binding domain that interacts with Rac and plays a role incellular transformation through the Ras-MAPK pathway; and murine NIK,which interacts with upstream receptor tyrosine kinases and connectswith downstream STE11-family kinases.

The STE20-kinases possess a variety of non-catalytic domains that arebelieved to interact with upstream regulators. Examples includeproline-rich domains for interaction with SH3-containing proteins, orspecific domains for interaction with Rac, Rho, and Rab smallG-proteins. These interactions may provide a mechanism for cross-talkbetween distinct biochemical pathways in response to external stimulisuch as the activation of a variety of cell surface receptors, includingtyrosine kinases, cytokine receptors, TNF receptor, Fas, T cellreceptors, CD28, or CD40.

I. The Nucleic Acids of the Invention

Included within the scope of this invention are the functionalequivalents of the herein-described isolated nucleic acid molecules. Thedegeneracy of the genetic code permits substitution of certain codons byother codons that specify the same amino acid and hence would give riseto the same protein. The nucleic acid sequence can vary substantiallysince, with the exception of methionine and tryptophan, the known aminoacids can be coded for by more than one codon. Thus, portions or all ofthe kinase genes of the invention could be synthesized to give a nucleicacid sequence significantly different from that shown in SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, andSEQ ID NO:106. The encoded amino acid sequence thereof would, however,be preserved.

In addition, the nucleic acid sequence may comprise a nucleotidesequence which results from the addition, deletion or substitution of atleast one nucleotide to the 5′-end and/or the 3′-end of the nucleic acidformula shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ IDNO:102, SEQ ID NO:104, or SEQ ID NO:106, or a derivative thereof. Anynucleotide or polynucleotide may be used in this regard, provided thatits addition, deletion or substitution does not alter the amino acidsequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:29, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ IDNO:103, SEQ ID NO:105, or SEQ ID NO:107, which is encoded by thenucleotide sequence. For example, the present invention is intended toinclude any nucleic acid sequence resulting from the addition of ATG asan initiation codon at the 5′-end of the inventive nucleic acid sequenceor its derivative, or from the addition of TTA, TAG or TGA as atermination codon at the 3′-end of the inventive nucleotide sequence orits derivative. Moreover, the nucleic acid molecule of the presentinvention may, as necessary, have restriction endonuclease recognitionsites added to its 5′-end and/or 3′-end.

Such functional alterations of a given nucleic acid sequence afford anopportunity to promote secretion and/or processing of heterologousproteins encoded by foreign nucleic acid sequences fused thereto. Allvariations of the nucleotide sequence of the kinase genes of theinvention and fragments thereof permitted by the genetic code are,therefore, included in this invention.

Further, it is possible to delete codons or to substitute one or morecodons with codons other than degenerate codons to produce astructurally modified polypeptide, but one which has substantially thesame utility or activity as the polypeptide produced by the unmodifiednucleic acid molecule. As recognized in the art, the two polypeptidesare functionally equivalent, as are the two nucleic acid molecules thatgive rise to their production, even though the differences between thenucleic acid molecules are not related to the degeneracy of the geneticcode.

Mammalian STLK2

The full-length human STLK2 cDNA (SEQ ID NO:1) is 3268 bp long andconsists of a 1248 bp open reading frame (ORF) flanked by a 181 bp 5′untranslated region (UTR; 1-181) and a 1784 bp 3′ UTR (1433-3216) thatis followed by a 52 nucleotide polyadenylated region. A polyadenylationsignal (AATAAA) is found at positions (3193-3198). The sequence flankingthe first ATG conforms to the Kozak consensus (Kozak, M., Nucleic AcidsRes. 15, 8125-8148 (1987)) for an initiating methionine, and is believedto be the translational start site for STLK2. Furthermore, human STLK2and the related SOK-1 and MST3 proteins conserve the amino acid sequenceimmediately following this presumed initiating methionine.

Several EST fragments span the complete STLK2 sequence with AA191319 atthe 5′ end and W16504 at the 3′ end.

Mammalian STLK3

The partial human STLK3 cDNA (SEQ ID NO:2) is 3030 bp long and consistsof a 1548 bp ORF flanked by a 1476 bp 3′ UTR (1550-3025) and a 5nucleotide polyadenylated region. A potential polyadenylation signal(AATAAA) begins at position 3004. Since the coding region is openthroughout the 5′ extent of this sequence, this is apparently a partialcDNA clone lacking the N-terminal start methionine.

Multiple EST fragments span the complete STLK3 sequence with AA278967 atthe 5′ end and AA628477 and others at the 3′ end.

Mammalian STLK4

The partial human STLK4 cDNA (SEQ ID NO:3) is 3857 bp long and consistsof a 1242 bp ORF flanked by a 2596 bp 3′ UTR (1244-3839) and an 18nucleotide polyadenylated region. A potential polyadenylation signal(AATAAA) is found at positions 2181-3822. Since the coding region isopen throughout the 5′ extent of this sequence, this is apparently apartial cDNA clone lacking the N-terminal start methionine. A nearfull-length murine STLK4 cDNA is represented in the 1773 bp ESTAA117438. It extends an additional 21 nucleotides 5′ of the human STLK4consensus, but since its coding region is open throughout the 5′ extentof the sequence, this is also apparently a partial cDNA clone lackingthe N-terminal start methionine.

Several EST fragments span the complete STLK3 sequence with AA297759 atthe 5′ end and AA100484 and others at the 3′ end.

Mammalian STLK5

The full-length human STLK5 cDNA (SEQ ID NO:96) is 2110 bp long andconsists of a 1119 bp ORF flanked by a 229 bp 5′ UTR and a 762 bp 3′UTR. The sequence flanking the first ATG conforms to the Kozak consensus(supra) for an initiating methionine, and is believed to be thetranslational start site for STLK5. Several EST fragments span thecomplete STLK5 sequence with AA297059 and F07734 at the 5′ end, andR46686 and F03423 and others at the 3′ end.

Mammalian STLK6

The full-length human STLK6 cDNA (SEQ ID NO:98) is 2,001 bp long andconsists of a 1,254 bp ORF flanked by a 75 bp 5′ UTR and a 673 bp 3′UTR. The sequence flanking the first ATG conforms to the Kozak consensus(supra) for an initiating methionine, and is believed to be thetranslational start site for STLK6.

Mammalian STLK7

The partial human STLK7 cDNA (SEQ ID NO:100) is 311 bp long and consistsof a 309 bp ORF. Since the coding region is open throughout both the 5′and 3′ extent of this sequence, this is apparently a partial cDNA clonelacking the N-terminal start methionine and C-terminal stop codon.

Mammalian ZC1

The full-length human ZC1 cDNA (SEQ ID NO:9) is 3798 bp long andconsists of a 3717 bp ORF (7-3723) flanked by a 6 bp 5′ UTR and a 75 bp(3724-3798) 3′ UTR. No polyadenylation signal (AATAAA) or polyadenylatedregion are present in the 3′UTR. The sequence flanking the first ATGconforms to the Kozak consensus for an initiating methionine, and isbelieved to be the translational start site for human ZC1.

Multiple EST fragments (W81656) match the 3′ end of the human ZC1 gene,but at the time of filing, the inventors believe that none exist inGenBank or the EST database that match its 5′ end.

Mammalian ZC2

The partial human ZC2 cDNA (SEQ ID NO:10) is 4055 bp long and consistsof a 3891 bp ORF (1-3891) and a 164 bp (3892-4055) 3′ UTR. Since thecoding region is open throughout the 5′ extent of this sequence, this isapparently a partial cDNA clone lacking the N-terminal start methionine.No polyadenylation signal (AATAAA) or polyadenylated region are presentin the 3′UTR.

Multiple EST fragments (R51245) match the 3′ end of the human ZC2 gene,but at the time of filing, the inventors believe that none exist inGenBank or the EST database that match its 5′ end.

Mammalian ZC3

The partial human ZC3 cDNA (SEQ ID NO:11) is 4133 bp long and consistsof a 3978 bp ORF (1-3978) and a 152 bp (3979-4133) 3′UTR region. Sincethe coding region is open throughout the 5′ extent of this sequence,this is apparently a partial cDNA clone lacking the N-terminal startmethionine. No polyadenylation signal (AATAAA) or polyadenylated regionare present in the 3′UTR.

Multiple EST fragments (R54563) match the 3′end of the human ZC3 gene,but at the time of filing, the inventors believe that none exist inGenBank or the EST database that match its 5′ end.

Mammalian ZC4

The full-length human ZC4 cDNA (SEQ ID NO:104) is 3,684 bp long and wasoriginally assembled from X chromosome genomic DNA sequence.

Multiple EST fragments (R98571) match the 3′end of the human ZC4 gene,but at the time of filing, the inventors believe that none exist inGenBank or the EST database that match its 5′ end. ZC4 gene is alsocontained within the human genomic clone Z83850.

Mammalian KHS2

The full-length human KHS2 cDNA (SEQ ID NO:17) is 4023 bp long andconsists of a 2682 bp ORF (6-2687) flanked by a 5 bp (1-5) 5′UTR and a1336 bp (2688-4023) 3′ UTR. A potential polyadenylation signal (AATAAA)is found at positions 4008-4013. No polyadenylated region is present inthe 3′UTR. The sequence flanking the first ATG conforms to the Kozakconsensus for an initiating methionine, and is believed to be thetranslational start site for human KHS2.

Multiple EST fragments match the 5′end (AA446022) as well as the 3′ end(R37625) of the human KHS2 gene.

Mammalian SULU1

The full-length human SULU1 cDNA (SEQ ID NO:19) is 4177 bp long andconsists of a 2694 bp ORF (415-3108) flanked by a 414 bp (1-414) 5′UTRand a 1069 bp (3109-4177) 3′ UTR followed by a 19 nucleotidepolydenylated region. A potential polyadenylation signal (AATAAA) isfound at positions 4164-4169. The sequence flanking the first ATGconforms to the Kozak consensus for an initiating methionine, and isbelieved to be the translational start site for human SULU1.

Multiple EST fragments match the 5′end (N27153) as well as the 3′ end(R90908) of the human SULU1 gene.

Mammalian (Murine) SULU3

The partial murine SULU3 cDNA (SEQ ID NO:21) is 2249 bp long andconsists of a 2244 bp ORF (6-2249) flanked by a 5 bp (1-5) 5′UTR. Thesequence flanking the first ATG conforms to the Kozak consensus for aninitiating methionine, and is believed to be the translational startsite for murine SULU3. The 3′ end of the murine SULU3 cDNA shares 90%DNA sequence identity over 1620 nucleotides with human SULU3, suggestingthat these two genes are functional orthologues.

One EST fragment (AA446022) matches the 3′ end of the partial murineSULU3 gene, but at the time of filing, the inventors believe that noneexist in GenBank or the EST database that match its 5′ end.

Mammalian (Human) SULU3

The partial human SULU3 cDNA (SEQ ID NO:20) is 3824 bp long and consistsof a 2358 bp ORF (2-2359) flanked by a 1465 bp (2360-3824) 3′UTRfollowed by a 19 nucleotide polydenylated region. A potentialpolyadenylation signal (AATAAA) is found at positions 2602-2607. Sincethe coding region is open throughout the 5′ extent of this sequence,this is apparently a partial cDNA clone lacking the N-terminal startmethionine. The 5′ end of the human SULU3 cDNA shares 90% DNA sequenceidentity over 1620 nucleotides with murine SULU3, suggesting that thesetwo genes are functional orthologues.

Multiple EST fragments (R02283) match the 3′end of the human SULU3 gene,but at the time of filing, the inventors believe that none exist inGenBank or the EST database that match its 5′ end.

Mammalian GEK2

The full-length human GEK2 cDNA (SEQ ID NO:106) is 2962 bp long andconsists of a 2737 bp ORF (59-2795) flanked by a 58 bp (1-58) 5′UTR. Thesequence flanking the first ATG conforms to the Kozak consensus for aninitiating methionine, and is believed to be the translational startsite for human GEK2.

Multiple EST fragments (AA465671) match the 5′end, but at the time offiling, the inventors believe that only one (AA380492) matches the 3′end of the human GEK2 gene.

Mammalian PAK4

The full-length human PAK4 cDNA (SEQ ID NO:27) is 3604 bp long andconsists of a 2043 bp ORF (143-2185) flanked by a 142 bp (1-142) 5′UTRand a 1419 3′ UTR followed by a 22 nucleotide polydenylated region. Apotential polyadenylation signal (AATTAAA) is found at positions3582-3588. The sequence flanking the first ATG conforms to the Kozakconsensus for an initiating methionine, and is believed to be thetranslational start site for human PAK4.

Multiple EST fragments (AA535791) match the 3′end of the human PAK4gene, but at the time of filing, the inventors believe that none existin GenBank or the EST database that match its 5′ end.

Mammalian PAK5

The full-length human PAK5 cDNA (SEQ ID NO:102) is 2806 bp long andconsists of a 1773 bp ORF flanked by a 201 bp 5′ UTR and a 833 bp 3′UTR. The sequence flanking the first ATG conforms to the Kozak consensus(supra) for an initiating methionine, and is believed to be thetranslational start site for PAK5.

Multiple EST fragments (AA442867) match the 3′end of the human PAK5gene, but at the time of filing, the inventors believe that none existin GenBank or the EST database that match its 5′ end.

II. Nucleic Acid Probes, Methods, and Kits for Detection ofSTE20-Related Kinases.

A nucleic acid probe of the present invention may be used to probe anappropriate chromosomal or cDNA library by usual hybridization methodsto obtain other nucleic acid molecules of the present invention. Achromosomal DNA or cDNA library may be prepared from appropriate cellsaccording to recognized methods in the art (cf. “Molecular Cloning: ALaboratory Manual”, second edition, Cold Spring Harbor Laboratory,Sambrook, Fritsch, & Maniatis, eds., 1989).

In the alternative, chemical synthesis can be carried out in order toobtain nucleic acid probes having nucleotide sequences which correspondto N-terminal and C-terminal portions of the amino acid sequence of thepolypeptide of interest. The synthesized nucleic acid probes may be usedas primers in a polymerase chain reaction (PCR) carried out inaccordance with recognized PCR techniques, essentially according to PCRProtocols, “A Guide to Methods and Applications”, Academic Press,Michael, et al., eds., 1990, utilizing the appropriate chromosomal orcDNA library to obtain the fragment of the present invention.

One skilled in the art can readily design such probes based on thesequence disclosed herein using methods of computer alignment andsequence analysis known in the art (“Molecular Cloning: A LaboratoryManual”, 1989, supra). The hybridization probes of the present inventioncan be labeled by standard labeling techniques such as with aradiolabel, enzyme label, fluorescent label, biotin-avidin label,chemiluminescence, and the like. After hybridization, the probes may bevisualized using known methods.

The nucleic acid probes of the present invention include RNA, as well asDNA probes, such probes being generated using techniques known in theart. The nucleic acid probe may be immobilized on a solid support.Examples of such solid supports include, but are not limited to,plastics such as polycarbonate, complex carbohydrates such as agaroseand sepharose, and acrylic resins, such as polyacrylamide and latexbeads. Techniques for coupling nucleic acid probes to such solidsupports are well known in the art.

The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The samples used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample which is compatiblewith the method utilized.

One method of detecting the presence of nucleic acids of the inventionin a sample comprises (a) contacting said sample with theabove-described nucleic acid probe under conditions such thathybridization occurs, and (b) detecting the presence of said probe boundto said nucleic acid molecule. One skilled in the art would select thenucleic acid probe according to techniques known in the art as describedabove. Samples to be tested include but should not be limited to RNAsamples of human tissue.

A kit for detecting the presence of nucleic acids of the invention in asample comprises at least one container means having disposed thereinthe above-described nucleic acid probe. The kit may further compriseother containers comprising one or more of the following: wash reagentsand reagents capable of detecting the presence of bound nucleic acidprobe. Examples of detection reagents include, but are not limited toradiolabelled probes, enzymatic labeled probes (horseradish peroxidase,alkaline phosphatase), and affinity labeled probes (biotin, avidin, orsteptavidin).

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allow the efficient transfer of reagents from one compartmentto another compartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the probe or primers used in the assay,containers which contain wash reagents (such as phosphate bufferedsaline, Tris-buffers, and the like), and containers which contain thereagents used to detect the hybridized probe, bound antibody, amplifiedproduct, or the like. One skilled in the art will readily recognize thatthe nucleic acid probes described in the present invention can readilybe incorporated into one of the established kit formats which are wellknown in the art.

III. DNA Constructs Comprising a STE20-Related Nucleic Acid Molecule andCells Containing These Constructs.

The present invention also relates to a recombinant DNA moleculecomprising, 5′ to 3′, a promoter effective to initiate transcription ina host cell and the above-described nucleic acid molecules. In addition,the present invention relates to a recombinant DNA molecule comprising avector and an above-described nucleic acid molecule. The presentinvention also relates to a nucleic acid molecule comprising atranscriptional region functional in a cell, a sequence complementary toan RNA sequence encoding an amino acid sequence corresponding to theabove-described polypeptide, and a transcriptional termination regionfunctional in said cell. The above-described molecules may be isolatedand/or purified DNA molecules.

The present invention also relates to a cell or organism that containsan above-described nucleic acid molecule and thereby is capable ofexpressing a polypeptide. The polypeptide may be purified from cellswhich have been altered to express the polypeptide. A cell is said to be“altered to express a desired polypeptide” when the cell, throughgenetic manipulation, is made to produce a protein which it normallydoes not produce or which the cell normally produces at lower levels.One skilled in the art can readily adapt procedures for introducing andexpressing either genomic, cDNA, or synthetic sequences into eithereukaryotic or prokaryotic cells.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene sequence expression. Theprecise nature of the regulatory regions needed for gene sequenceexpression may vary from organism to organism, but shall in generalinclude a promoter region which, in prokaryotes, contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which, when transcribed into RNA, will signalsynthesis initiation. Such regions will normally include those5′-non-coding sequences involved with initiation of transcription andtranslation, such as the TATA box, capping sequence, CAAT sequence, andthe like.

If desired, the non-coding region 3′ to the sequence encoding a kinaseof the invention may be obtained by the above-described methods. Thisregion may be retained for its transcriptional termination regulatorysequences, such as termination and polyadenylation. Thus, by retainingthe 3′-region naturally contiguous to the DNA sequence encoding a kinaseof the invention, the transcriptional termination signals may beprovided. Where the transcriptional termination signals are notsatisfactorily functional in the expression host cell, then a 3′ regionfunctional in the host cell may be substituted.

Two DNA sequences (such as a promoter region sequence and a sequenceencoding a kinase of the invention) are said to be operably linked ifthe nature of the linkage between the two DNA sequences does not (1)result in the introduction of a frame-shift mutation, (2) interfere withthe ability of the promoter region sequence to direct the transcriptionof a gene sequence encoding a kinase of the invention, or (3) interferewith the ability of the gene sequence of a kinase of the invention to betranscribed by the promoter region sequence. Thus, a promoter regionwould be operably linked to a DNA sequence if the promoter were capableof effecting transcription of that DNA sequence. Thus, to express a geneencoding a kinase of the invention, transcriptional and translationalsignals recognized by an appropriate host are necessary.

The present invention encompasses the expression of a gene encoding akinase of the invention (or a functional derivative thereof) in eitherprokaryotic or eukaryotic cells. Prokaryotic hosts are, generally, veryefficient and convenient for the production of recombinant proteins andare, therefore, one type of preferred expression system for kinases ofthe invention. Prokaryotes most frequently are represented by variousstrains of E. coli. However, other microbial strains may also be used,including other bacterial strains.

In prokaryotic systems, plasmid vectors that contain replication sitesand control sequences derived from a species compatible with the hostmay be used. Examples of suitable plasmid vectors may include pBR322,pUC118, pUC119 and the like; suitable phage or bacteriophage vectors mayinclude γgt10, γgt11 and the like; and suitable virus vectors mayinclude pMAM-neo, pKRC and the like. Preferably, the selected vector ofthe present invention has the capacity to replicate in the selected hostcell.

Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus,Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However,under such conditions, the polypeptide will not be glycosylated. Theprokaryotic host must be compatible with the replicon and controlsequences in the expression plasmid.

To express a kinase of the invention (or a functional derivativethereof) in a prokaryotic cell, it is necessary to operably link thesequence encoding the kinase of the invention to a functionalprokaryotic promoter. Such promoters may be either constitutive or, morepreferably, regulatable (i.e., inducible or derepressible). Examples ofconstitutive promoters include the int promoter of bacteriophage λ, thebla promoter of the β-lactamase gene sequence of pBR322, and the catpromoter of the chloramphenicol acetyl transferase gene sequence ofpPR325, and the like. Examples of inducible prokaryotic promotersinclude the major right and left promoters of bacteriophage λ (P_(L) andP_(R)), the trp, recA, λacZ, λacI, and gal promoters of E. coli, theα-amylase (Ulmanen et al., J. Bacteriol. 162: 176-182, 1985) and thec-28-specific promoters of B. subtilis (Gilman et al., Gene Sequence 32:11-20, 1984), the promoters of the bacteriophages of Bacillus (Gryczan,In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY,1982), and Streptomyces promoters (Ward et al., Mol. Gen. Genet. 203:468-478, 1986). Prokaryotic promoters are reviewed by Glick (Ind.Microbiot. 1: 277-282, 1987), Cenatiempo (Biochimie 68: 505-516, 1986),and Gottesman (Ann. Rev. Genet. 18: 415-442, 1984).

Proper expression in a prokaryotic cell also requires the presence of aribosome-binding site upstream of the gene sequence-encoding sequence.Such ribosome-binding sites are disclosed, for example, by Gold et al.(Ann. Rev. Microbiol. 35: 365-404, 1981). The selection of controlsequences, expression vectors, transformation methods, and the like, aredependent on the type of host cell used to express the gene. As usedherein, “cell”, “cell line”, and “cell culture” may be usedinterchangeably and all such designations include progeny. Thus, thewords “transformants” or “transformed cells” include the primary subjectcell and cultures derived therefrom, without regard to the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.However, as defined, mutant progeny have the same functionality as thatof the originally transformed cell.

Host cells which may be used in the expression systems of the presentinvention are not strictly limited, provided that they are suitable foruse in the expression of the kinase polypeptide of interest. Suitablehosts may often include eukaryotic cells. Preferred eukaryotic hostsinclude, for example, yeast, fungi, insect cells, mammalian cells eitherin vivo, or in tissue culture. Mammalian cells which may be useful ashosts include HeLa cells, cells of fibroblast origin such as VERO orCHO-K1, or cells of lymphoid origin and their derivatives. Preferredmammalian host cells include SP2/0 and J558L, as well as neuroblastomacell lines such as IMR 332, which may provide better capacities forcorrect post-translational processing.

In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thecauliflower mosaic virus 35S and 19S, and nopaline synthase promoter andpolyadenylation signal sequences. Another preferred host is an insectcell, for example the Drosophila larvae. Using insect cells as hosts,the Drosophila alcohol dehydrogenase promoter can be used (Rubin,Science 240: 1453-1459, 1988). Alternatively, baculovirus vectors can beengineered to express large amounts of kinases of the invention ininsect cells (Jasny, Science 238: 1653, 1987; Miller et al., In: GeneticEngineering, Vol. 8, Plenum, Setlow et al., eds., pp. 277-297, 1986).

Any of a series of yeast expression systems can be utilized whichincorporate promoter and termination elements from the activelyexpressed sequences coding for glycolytic enzymes that are produced inlarge quantities when yeast are grown in mediums rich in glucose. Knownglycolytic gene sequences can also provide very efficienttranscriptional control signals. Yeast provides substantial advantagesin that it can also carry out post-translational modifications. A numberof recombinant DNA strategies exist utilizing strong promoter sequencesand high copy number plasmids which can be utilized for production ofthe desired proteins in yeast. Yeast recognizes leader sequences oncloned mammalian genes and secretes peptides bearing leader sequences(i.e., pre-peptides). Several possible vector systems are available forthe expression of kinases of the invention in a mammalian host.

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus,cytomegalovirus, simian virus, or the like, where the regulatory signalsare associated with a particular gene sequence which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, and the like, may be employed.Transcriptional initiation regulatory signals may be selected whichallow for repression or activation, so that expression of the genesequences can be modulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

Expression of kinases of the invention in eukaryotic hosts requires theuse of eukaryotic regulatory regions. Such regions will, in general,include a promoter region sufficient to direct the initiation of RNAsynthesis. Preferred eukaryotic promoters include, for example, thepromoter of the mouse metallothionein I gene sequence (Hamer et al., J.Mol. Appl. Gen. 1: 273-288, 1982); the TK promoter of Herpes virus(McKnight, Cell 31: 355-365, 1982); the SV40 early promoter (Benoist etal., Nature (London) 290: 304-31, 1981); and the yeast gal4 genesequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982; Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984).

Translation of eukaryotic mRNA is initiated at the codon which encodesthe first methionine. For this reason, it is preferable to ensure thatthe linkage between a eukaryotic promoter and a DNA sequence whichencodes a kinase of the invention (or a functional derivative thereof)does not contain any intervening codons which are capable of encoding amethionine (i.e., AUG). The presence of such codons results either inthe formation of a fusion protein (if the AUG codon is in the samereading frame as the kinase of the invention coding sequence) or aframe-shift mutation (if the AUG codon is not in the same reading frameas the kinase of the invention coding sequence).

A nucleic acid molecule encoding a kinase of the invention and anoperably linked promoter may be introduced into a recipient prokaryoticor eukaryotic cell either as a nonreplicating DNA or RNA molecule, whichmay either be a linear molecule or, more preferably, a closed covalentcircular molecule. Since such molecules are incapable of autonomousreplication, the expression of the gene may occur through the transientexpression of the introduced sequence. Alternatively, permanentexpression may occur through the integration of the introduced DNAsequence into the host chromosome.

A vector may be employed which is capable of integrating the desiredgene sequences into the host cell chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes can be selected byalso introducing one or more markers which allow for selection of hostcells which contain the expression vector. The marker may provide forprototrophy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene sequence can either be directly linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection. Additional elements may also be needed for optimalsynthesis of mRNA. These elements may include splice signals, as well astranscription promoters, enhancers, and termination signals. cDNAexpression vectors incorporating such elements include those describedby Okayama (Mol. Cell. Biol. 3: 280-, 1983).

The introduced nucleic acid molecule can be incorporated into a plasmidor viral vector capable of autonomous replication in the recipient host.Any of a wide variety of vectors may be employed for this purpose.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coli (such as, for example, pBR322, Co1E1, pSC101,pACYC 184, □VX; “Molecular Cloning: A Laboratory Manual”, 1989, supra).Bacillus plasmids include pC194, pC221, pT127, and the like (Gryczan,In: The Molecular Biology of the Bacilli, Academic Press, NY, pp.307-329, 1982). Suitable Streptomyces plasmids include p1J101 (Kendallet al., J. Bacteriol. 169: 4177-4183, 1987), and streptomycesbacteriophages such as □C31 (Chater et al., In: Sixth InternationalSymposium on Actinomycetales Biology, Akademiai Kaido, Budapest,Hungary, pp. 45-54, 1986). Pseudomonas plasmids are reviewed by John etal. (Rev. Infect. Dis. 8: 693-704, 1986), and Izaki (Jpn. J. Bacteriol.33: 729-742, 1978).

Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40,2-micron circle, and the like, or their derivatives. Such plasmids arewell known in the art (Botstein et al., Miami Wntr. Symp. 19: 265-274,1982; Broach, In: “The Molecular Biology of the Yeast Saccharomyces:Life Cycle and Inheritance”, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., p. 445-470, 1981; Broach, Cell 28: 203-204, 1982; Bollonet al., J. Clin. Hematol. Oncol. 10: 39-48, 1980; Maniatis, In: CellBiology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression,Academic Press, NY, pp. 563-608, 1980).

Once the vector or nucleic acid molecule containing the construct(s) hasbeen prepared for expression, the DNA construct(s) may be introducedinto an appropriate host cell by any of a variety of suitable means,i.e., transformation, transfection, conjugation, protoplast fusion,electroporation, particle gun technology, calciumphosphate-precipitation, direct microinjection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene(s) results in the production of a kinaseof the invention, or fragments thereof. This can take place in thetransformed cells as such, or following the induction of these cells todifferentiate (for example, by administration of bromodeoxyuracil toneuroblastoma cells or the like). A variety of incubation conditions canbe used to form the peptide of the present invention. The most preferredconditions are those which mimic physiological conditions.

IV. The Proteins of the Invention

A variety of methodologies known in the art can be utilized to obtainthe polypeptides of the present invention. The polypeptides may bepurified from tissues or cells that naturally produce the polypeptides.Alternatively, the above-described isolated nucleic acid fragments couldbe used to express the kinases of the invention in any organism. Thesamples of the present invention include cells, protein extracts ormembrane extracts of cells, or biological fluids. The samples will varybased on the assay format, the detection method, and the nature of thetissues, cells or extracts used as the sample.

Any eukaryotic organism can be used as a source for the polypeptides ofthe invention, as long as the source organism naturally contains suchpolypeptides. As used herein, “source organism” refers to the originalorganism from which the amino acid sequence of the subunit is derived,regardless of the organism the subunit is expressed in and ultimatelyisolated from.

One skilled in the art can readily follow known methods for isolatingproteins in order to obtain the polypeptides free of naturalcontaminants. These include, but are not limited to: size-exclusionchromatography, HPLC, ion-exchange chromatography, and immuno-affinitychromatography.

Mammalian STLK2

Analysis of the deduced amino acid sequence predicts STLK2 to be anintracellular serine/threonine kinase, lacking both a signal sequenceand transmembrane domain. STLK2 contains a 21 amino acid N-terminaldomain, a 253 amino acid catalytic domain with all the motifscharacteristic of a serine/threonine kinase, followed by a 142 aminoacid C-terminal domain.

STLK2 is most closely related to human STE20-subfamily kinases, MST3(GB:AF024636) and SOK-1 (GB:X99325) and a C. elegans kinase yk34b11.5(GB:U53153) sharing 72.7%, 68.7%, and 69.3% amino acid identity,respectively.

The 21 amino acid N-terminal domain of human STLK2 is 71.4% identical tothe N-terminus of MST3 (GB:AF024636). Human STLK2 lacks a glycineresidue at position 2, and is therefore unlikely to undergomyristylation. A Smith-Waterman search of the nonredundant proteindatabase does not reveal any significant homologies that might suggest apotential function for this domain.

The 253 amino acid catalytic domain of human STLK2 is most related tohuman SOK-1 (X99325), MST3 (GB:AF024636), C. elegans yk32b11.5(GB:U53153), and STLK3 (SEQ ID NO:6) sharing 88.9%, 87.4%, 78.3%, and49% amino identity respectively, placing it in the STLK-subfamily ofSTE20-related kinases. The STLK2 kinase domain displayed lesser homologyto other STE20-related kinases including: 55.9% to human MST2(GB:U26424), 49.2% to human GCK (GB:U07349), 49.2% to human KHS1(GB:U77129), and 44.2% to human HPK1 (GB:U66464). The activation loop ofhuman STLK2 catalytic domain is identical to that of human SOK-1 andMST3 including the presence of four potential threonine phosphorylationsites that could serve an autoregulatory role on kinase activity.

The 142 amino acid C-terminal domain of human STLK2 is most related tohuman SOK-1 (X99325), MST3 (GB:AF024636), and C. elegans yk32b11.5(GB:U53153), sharing 39.9%, 39.9%, and 33.3% amino acid identity,respectively. This C-terminal domain shares some significant amino acidsimilarity to the C-terminal domains of the related human STLK3 (SEQ IDNO:6) and STLK4 (SEQ ID NO:7).

The C-terminus of the related human SOK-1 (GB:X99325) kinase has beenshown to be inhibitory to the catalytic activity of this kinase (Pombo,C. M., Bonventre, J. V., Molnar, A., Kyriakis, J. and Force, T. EMBO J.15, 4537-4546 (1996)). Based on the sequence identity between theC-termini of human SOK-1 (GB:X99325) and human STLK2 (39.2%), theC-terminus of human STLK2 may also function as an inhibitory domain forits kinase.

Mammalian STLK3

The 3030 bp human STLK3 nucleotide sequence of the partial cDNA cloneencodes a polypeptide of 516 amino acids (SEQ ID NO:6) with a predictedmolecular mass of 56,784 daltons. Analysis of the deduced amino acidsequence predicts STLK3 to be an intracellular serine/threonine kinase,lacking both a signal sequence and transmembrane domain, however thecDNA clone lacks an initiating ATG, so the full extent of it N-terminusis not known. STLK3 contains a 31 amino acid N-terminal domain, a 277amino acid catalytic domain with all the motifs characteristic of aserine/threonine kinase, followed by a 181 amino acid C-terminal domaincontaining a 25 amino acid insert and a 27 amino acid tail relative tothe sequence of human STLK2.

STLK3 is most closely related to human STE20-subfamily kinases, STLK4(SEQ ID. NO:7), MST3 (GB:AF024636), SOK-1 (GB:X99325) and STLK2 (SEQ IDNO:5) sharing 71.1%, 37.6%, 38.1%, and 38.4% amino acid identityrespectively.

The 31 amino acid N-terminal domain of human STLK3 lacked anysignificant amino acid sequence homologies using a Smith-Waterman searchof the nonredundant protein database, other than sequence similarity toproline-alanine repeats.

The 277 amino acid catalytic domain of human STLK3 is most related tohuman STLK4 (SEQ ID NO:7), SOK-1 (GB:X99325), MST3 (GB:AF024636), andSTLK2 (SEQ ID NO:5) sharing 88.2%, 49.2%, 49%, and 49% amino acididentity, respectively. It also shares strong homology to other STKsfrom lower organisms including 51.7% to A. thaliana (GB: AC002343),43.1% to A. thaliana (GB: Z97336), 42.1% to A. thaliana (GB: U96613),and 43.3% to C. elegans (GB: U53153). The activation loop of the humanSTLK3 catalytic domain conserves three potential threoninephosphorylation sites with other members of the STLK-subfamily ofSTE20-related kinases (human STE20, MST3, STLK2, STLK4) that could servean autoregulatory role on kinase activity.

The 181 amino acid C-terminal domain of human STLK3 shares 55.5% aminoacid identity to human STLK4 (SEQ ID NO:7), and is 100% identical to apartial human cDNA DCHT (GB:AF017635). The C-terminal domain of humanSTLK3 contains a 26 amino acid insert relative to human STE20. A similar(87.5% amino acid identity) 26 amino acid insert is also present inhuman STLK4.

The 27 amino acid C-terminal tail of human STLK3 shares 77.8% amino acididentity to human STLK4, but is absent from other STLK-family members.This high degree of homology between the C-tail of two STLK-familymembers suggests they may be involved in an as yet unidentifiedprotein-protein interaction.

The weak sequence homology between the C-termini of human STLK3 andSTE20, suggests it may also function as an inhibitory domain for itskinase.

Mammalian STLK4

The 3857 bp human STLK4 nucleotide sequence of the partial cDNA cloneencodes a polypeptide of 414 amino acids (SEQ ID NO:7) with a predictedmolecular mass of 45,451 daltons. Analysis of the deduced amino acidsequence predicts STLK4 to be an intracellular serine/threonine kinase,lacking both a signal sequence and transmembrane domain, however thecDNA clone lacks an initiating ATG, so the full extent of it N-terminusis not known. The partial STLK4 protein sequence contains a 178 aminoacid catalytic domain corresponding to the C-terminal motifs VI-XI of aserine/threonine kinase, followed by a 236 amino acid C-terminal domaincontaining two inserts of 25 and 41 amino acids each, relative to thesequence of human STLK2.

STLK4 is most closely related to human STE20-subfamily kinases, STLK3(SEQ ID. NO 6), MST3 (GB:AF024636), STLK2 (SEQ ID NO:5), and SOK-1(GB:X99325) sharing 71.0%, 46.8%, 43.9%, and 37.7% amino acid identity,respectively.

The 178 amino acid catalytic domain of human STLK4 is most related tohuman STLK3 (SEQ ID NO. 7), SOK-1 (GB:X99325), MST3 (GB:AF024636), STLK2(SEQ ID NO:5), and MST1 (GB:U18297), sharing 88.2%, 54.2%, 54.0%, 53.7and 45.7% amino acid identity, respectively. It also shares stronghomology to other STKs from lower organisms including 56.9% to A.thaliana (GB: AC002343), 52.5% to C. elegans (GB: U53153), 46.2% to A.thaliana (GB: Z97336) and 45.7% to A. thaliana (GB: U96613). Theactivation loop of the human STLK4 catalytic domain conserves threepotential threonine phosphorylation sites with other members of theSTLK-subfamily of STE20-related kinases (human STE20, MST3, STLK2 andSTLK3) that could serve an autoregulatory role on kinase activity.

The 236 amino acid C-terminal domain of human STLK4 shares 58.1% aminoacid identity to both human STLK3 (SEQ ID NO:6) and to a partial humancDNA, DCHT (GB:AF017635). The C-terminal domain of human STLK4 containsa 25 amino acid insert relative to human SOK-1 and shares 87.5% aminoacid identity to an insert present in human STLK3.

The weak sequence homology between the C-termini of human STLK4 andSTE20, suggests it may also function as an inhibitory domain for itskinase.

Mammalian STLK5

The full-length 2110 bp human STLK5 cDNA encodes a polypeptide of 373amino acids (SEQ ID NO:97) with a predicted molecular mass of 41,700daltons. Analysis of the deduced amino acid sequence predicts STLK5 tobe an intracellular STE20-subfamily kinase, lacking both a signalsequence and transmembrane domain. STLK5 contains a 10 amino acidN-terminal domain, a 311 amino acid catalytic domain with all the motifscharacteristic of a serine/threonine kinase, and a 52 amino acidC-terminal domain.

STLK5 is most closely related to the human STE20-subfamily kinases STLK6(SEQ ID No. 99) and SPAK (AF099989), sharing 51% and 33% amino acididentity, respectively, over its full extent. It also shares significanthomology to database entries from Arabidopsis thaliana (GB:AC002343) andC. elegans (GB:AL023843, GB:AL023843).

The 10 amino acid N-terminal domain of human STLK5 does not reveal anysignificant homologies to the protein database.

The 311 amino acid catalytic domain of human STLK5 shares 51% and 34%identity to STLK6 and SPAK, respectively. The catalytic domain of STLK5contains a 45 amino acid insert between kinase subdomains X and XIrelative to human STE20. Multiple human EST fragments as well as amurine EST (GB:AA575647) contain this insert providing evidence thatthis region is an integral part of STLK5.

The 52 amino acid C-terminal tail of human STLK5 shares 41.3% amino acididentity to human SOK-1 (GB:X99325). The weak sequence homology betweenthe C-termini of human STLK5 and STE20, suggests it may also function asan inhibitory domain for its kinase.

Mammalian STLK6

The 2,001 bp human STLK6 nucleotide sequence of the complete cDNAencodes a polypeptide of 418 amino acids (SEQ ID NO:99) with a predictedmolecular mass of 47,025 daltons. Analysis of the deduced amino acidsequence predicts STLK6 to be an intracellular STE20-subfamily kinase,lacking both a signal sequence and transmembrane domain. STLK6 containsa 57 amino acid N-terminal domain, a 312 amino acid catalytic domainwith all the motifs characteristic of a serine/threonine kinase,followed by a 49 amino acid C-terminal domain.

STLK6 is most closely related to human STE20-subfamily kinases STLK5(SEQ ID NO:97), STLK7 (SEQ ID NO:101), and SPAK (AF099989), sharing 50%,35%, and 30% amino acid identity over its full extent. It also sharessignificant homology to database entries from Arabidopsis thaliana(GB:AC002343) and C. elegans (GB:U53153).

The 57 amino acid N-terminal domain of human STLK6 does not reveal anysignificant homologies in the protein database.

The 312 amino acid catalytic domain of human STLK6 shares 51 and 30%identity to human STLK5 and SPAK, respectively.

The 49 amino acid C-terminal tail of human STLK6 shares low amino acidsequence identity (29%) with STLK5 and SPAK.

Mammalian STLK7

The 311 bp human STLK7 nucleotide sequence of the partial cDNA encodes apolypeptide of 103 amino acids (SEQ ID NO:101). Analysis of the deducedamino acid sequence predicts STLK7 to be an internal fragment of anintracellular STE20-family kinase. This sequence lacks the N- andC-terminal portions of STLK7 and contains only the N-terminal 103 aminoacids of the predicted catalytic domain.

Human STLK7 is most closely related to human STE20-subfamily kinasesSPAK (AF099989), STLK5 (SEQ ID NO:97), and STLK6 (SEQ ID NO:99), sharing86%, 38%, and 35% amino acid identity within this region of the kinasedomain. It also shares significant homology to database entries fromArabidopsis thaliana (GB:AC002343) and Drosophila melanogaster(GB:AF006640).

Mammalian ZC1

The 3798 bp human ZC1 nucleotide sequence encodes a polypeptide of 1239amino acids (SEQ ID NO:13) with a predicted molecular mass of 142,140daltons. Analysis of the deduced amino acid sequence predicts ZC1 to bean intracellular serine/threonine kinase, lacking both a signal sequenceand transmembrane domain. The full-length ZC1 protein contains a 22amino acid N-terminus, a 267 amino acid catalytic domain with all themotifs characteristic of a serine/threonine kinase, a 237 amino acidregion predicted to form a coiled-coil structure, a 114 amino acidproline-rich region, a 256 amino acid spacer region, followed by a 343amino acid C-terminal domain containing a potential Rab/Rho-bindingregion.

ZC1 is most closely related to the human STE20-subfamily kinases ZC2(SEQ ID NO:14), ZC3 (SEQ ID NO:15), and ZC4 (SEQ ID NO:16), sharing61.7%, 60.9%, and 43.8% amino acid identity, respectively. ZC1 alsoshares 45.5% amino acid identity to a C. elegans kinase encoded by thecosmid ZC504.4 (GB:Z50029). ZC1 exhibits 90.0% amino acid homology tomurine NIK (GB:U88984), suggesting it may be the human orthologue ofthis STK.

The 22 amino acid N-terminal domain of human ZC1 is 58.8% identical tothe C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029), and100% identical to murine NIK (GB: U88984). Human ZC1 lacks a glycineresidue at position 2, and is therefore unlikely to undergomyristylation. A Smith-Waterman search of the nonredundant proteindatabase does not reveal any significant homologies that might suggest apotential function for this domain.

The 267 amino acid catalytic domain of human ZC1 is most related tohuman STE20-subfamily kinases, ZC3 (SEQ ID NO:15), ZC2 (SEQ ID NO:14),KHS2 (SEQ ID NO:18), SOK-1 (GB:X99325), GCK (GB:U07349), and GEK2 (SEQID NO:107), and to the C. elegans kinase encoded by the cosmid ZC504.4(GB:Z50029) sharing 90.6%, 90.2%, 50.6%, 47.4%, 45.4%, 42.5% and 82.6%amino acid identity, respectively. The ZC1 kinase domain shares 98.1%identity to murine NIK (GB:U88984). ZC1 contains the potential “TPY”regulatory phosphorylation site in its activation loop. This “TPY” motifis conserved in other STE20-related kinases, including ZC2, ZC3, ZC4,GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.

Immediately C-terminal to the kinase domain of human ZC1 is a 237 aminoacid region predicted to form a coiled-coil structure based on the Lupasalgorithm (Lupas, A. Meth. Enzymol. 266, 513-525 (1996)). This region ofZC1 is most related to human STE20-subfamily kinases, ZC3 (SEQ IDNO:15), ZC2 (SEQ ID NO:14), and GEK2 (SEQ ID NO:107), as well as tohuman PITSLRE (GB:U04824) sharing 65.5%, 65.4%, 25.3%, and 29.0% aminoacid identity, respectively. The ZC1 coiled-coil domain also shares90.6% amino acid homology to murine NIK. The C. elegans homologueZC504.4 shares 32.2% sequence identity over this region.

Within the predicted coiled-coil domain of human ZC1, and the relatedZC3, is a region predicted to form a leucine zipper(Leu-X6-Leu-X6-Leu-X6-Leu-X20-Leu-X6-Leu) (SEQ ID NO:149). The fact thatthis leucine repeat exists within a predicted coiled-coil structuresuggests that the leucine zipper may have a high probability of servingas a dimerization interface (Hirst, J. D. et al Protein Engineering 9657-662 (1996)) mediating a potential inter- or intra-moleculardimerization of human ZC1.

The 114 amino acid proline-rich region of human ZC1 is most related tohuman STE20-subfamily kinases, ZC2 (SEQ ID NO:14) and ZC3 (SEQ IDNO:15), sharing 35.8%, and 24.9%, respectively. The ZC1 proline-richdomain shares 36.4% amino acid homology to murine NIK (GB:U88984). Threepotential “PxxP” (SEQ ID NO: 148) SH3 domain-binding motifs (I, II andIII) are found within the proline-rich region of human ZC1. Motif I isconserved in human ZC1 and C. elegans ZC504.4 (GB:Z50029). Motif II isconserved in ZC1, ZC2, ZC3, ZC4 and C. elegans ZC504.4. Motif III isconserved in ZC1, ZC2, ZC3 and ZC4. Motifs II and III of murine NIK havebeen shown to bind the SH3 motif of the adaptor molecule Nck (Su, Y-C.et al, EMBO J. 16, 1279-1290 (1997)). From this evidence, human ZC1 mayhave the potential to bind to Nck or other SH3 or WW domain-containingproteins and participate in growth factor-induced signaling pathways.

The 256 amino acid spacer region of human ZC1 is most related to humanSTE20-subfamily kinases, ZC2 (SEQ ID NO:14) and ZC3 (SEQ ID NO:15), aswell as to human PITSLRE (GB:U04824), sharing 59.9%, 33.1%, 29.6%, and26.4% amino acid identity, respectively. It also shares 59.9% amino acidhomology to murine NIK. The C. elegans homologue ZC504.4 has onlylimited sequence similarity in this spacer region.

The 343 amino acid C-terminal of human ZC1 is most related to humanSTE20-subfamily kinases, ZC3 (SEQ ID NO:15), ZC2 (SEQ ID NO:14), and ZC4(SEQ ID NO:16), sharing 89.2%, 88.9%, and 42.3%, amino acid identity,respectively. The ZC1 C-terminal domain also shares 98.8% amino acididentity to murine NIK. The C. elegans homologue ZC504.4 also shares68.7% amino acid identity with the C-tail of human ZC1. A lower, yetsignificant, homology is also evident to human KHS2 (SEQ ID NO:18), GCK(GB:U07349), and murine citron (GB:U07349) with 26.6%, 23.1% and 36.2%amino acid identity, respectively. GCK is a STE20-family kinase whoseC-terminal domain has been shown to bind the small G-protein Rab8 (Ren,M. et al., Proc. Natl. Acad. Sci. 93, 5151-5155 (1996)). Citron is anon-kinase Rho-binding protein (Madaule, P. et al., FEBS Lett. 377,243-238 (1995)).

The sequence similarity of the C-terminal region of ZC1 to proteins thathave potential Rab- or Rho-binding domains suggests that ZC1 may signalthrough a small G-protein-dependant pathway.

Mammalian ZC2

The 4055 bp human ZC2 nucleotide sequence of the partial cDNA encodes apolypeptide of 1297 amino acids (SEQ ID NO:14) with a predictedmolecular mass of 147,785 daltons. Analysis of the deduced amino acidsequence predicts ZC2 to be an intracellular serine/threonine kinase,lacking both a signal sequence and transmembrane domain, however thecDNA clone lacks an initiating ATG, so the full extent of it N-terminusis not known. The N-terminally truncated ZC2 protein contains a 255amino acid catalytic domain with all the motifs characteristic of aserine/threonine kinase, a 187 amino acid region predicted to form acoiled-coil structure, a 184 amino acid proline-rich region, a 328 aminoacid spacer region, followed by a 343 amino acid C-terminal domaincontaining a potential Rab/Rho-binding region.

ZC2 is most closely related to the human STE20-subfamily kinases ZC3(SEQ ID NO:15), ZC1 (SEQ ID NO:13), and ZC4 (SEQ ID NO:16), sharing88.3%, 61.7%, and 41.9% amino acid identity, respectively, and shares41.7% amino acid identity to a C. elegans kinase encoded by the cosmidZC504.4 (GB:Z50029).

The 255 amino acid catalytic domain of human ZC2 is most related tohuman STE20-subfamily kinases, ZC1 (SEQ ID NO:13), ZC3 (SEQ ID NO:15),SOK-1 (GB:X99325), KHS2 (SEQ ID NO:18), MST1 (GB:U18297), and GCK(GB:U07349), and to the C. elegans kinase encoded by the cosmid ZC504.4(GB:Z50029) sharing 90.2%, 89.8%, 49.0%, 48.6%, 47.9%, 45.0 and 76.7%amino acid identity, respectively. ZC2 contains the potential “TPY”regulatory phosphorylation site in its activation loop. This “TPY” motifis conserved in other STE20-related kinases, including ZC1, ZC3, ZC4,GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.

Immediately C-terminal to the kinase domain of human ZC2 is a 187 aminoacid region predicted to form a coiled-coil structure based on the Lupasalgorithm (supra). This region of ZC2 is most related to humanSTE20-subfamily kinases, ZC1 (SEQ ID NO:13), ZC3 (SEQ ID NO:15), andGEK2 (SEQ ID NO:107), as well as to human PITSLRE (GB:U04824), sharing65.8%, 61.5%, 29.7% and 29.6% amino acid identity, respectively. The C.elegans homologue ZC504.4 shares 30.8% sequence identity over thisregion. Human ZC2 lacks the potential leucine zipper found in ZC1 as aconsequence of a 29 amino acid deletion relative to ZC1 and ZC3.

The 184 amino acid proline-rich region of human ZC2 is most related tohuman STE20-subfamily kinases, ZC3 (SEQ ID NO:15) and ZC1 (SEQ IDNO:13), sharing 35.9% and 28.6%, amino acid identity, respectively.Significant homology is also evident to the murine WW domain-bindingprotein WBP7 (GB:U92455), and to the human SH3 domain-binding protein3BP-1 (GB:X87671), with 27.7% and 25.3% amino acid identity,respectively.

ZC2 contains two of the potential “PxxP” (SEQ ID NO: 148) SH3domain-binding motifs (II and III) found within the proline-rich regionof human ZC1. Motif II is conserved in ZC1, ZC3, ZC4 and C. elegansZC504.4, and Motif III is conserved in ZC1, ZC3 and ZC4. Motifs II andIII of murine NIK have been shown to bind the SH3 motif of the adaptormolecule Nck. From this evidence, human ZC1 may have the potential tobind to Nck or other SH3 or WW domain-containing proteins, and toparticipate in growth factor-induced signaling pathways.

The 328 amino acid spacer region of human ZC2 is most related to humanSTE20-subfamily kinases ZC1 (SEQ ID NO:13) and ZC3 (SEQ ID NO:15), andto murine NIK (GB:U88984), sharing 31.6%, 26.9% and 25.9% amino acididentity, respectively. The C. elegans homologue ZC504.4 has onlylimited sequence similarity in this spacer region.

The 343 amino acid C-terminal of human ZC2 is most related to humanSTE20-subfamily kinases ZC1 (SEQ ID NO:13), ZC3 (SEQ ID NO:15) and ZC4(SEQ ID NO:16), and to murine NIK (GB:U88984), sharing 88.9%, 88.3%,41.9%, and 88.0%, amino acid identity, respectively. The C. eleganshomologue, ZC504.4, also shares 67.2% amino acid identity with theC-tail of human ZC2. A lower, yet significant, homology is also evidentto human GCK (GB:U07349), murine citron (GB:U07349), and the S.cerevisiae ROM2 protein (GB:U19103), a Rho1 GDP/GTP exchange factor,with 22.3%, 22.2% and 21.9% amino acid identity, respectively.

The sequence similarity of the C-terminal region of ZC2 to proteins thathave potential Rab- or Rho-binding domains suggests that ZC2, like ZC1,may also signal through a small G-protein-dependant pathway.

Mammalian ZC3

The 4133 bp human ZC3 nucleotide sequence of the partial cDNA encodes apolypeptide of 1326 amino acids (SEQ ID NO:15) with a predictedmolecular mass of 149,906 daltons. Analysis of the deduced amino acidsequence predicts ZC3 to be an intracellular serine/threonine kinase,lacking both a signal sequence and transmembrane domain, however thecDNA clone lacks an initiating ATG, so the full extent of it N-terminusis not known. The N-terminally truncated ZC3 protein contains a 255amino acid catalytic domain with all the motifs characteristic of aserine/threonine kinase: a 221 amino acid region predicted to form acoiled-coil structure, a 204 amino acid proline-rich region, and a 303amino acid spacer region followed by a 343 amino acid C-terminal domaincontaining a potential Rab/Rho-binding region.

ZC3 is most closely related to the human STE20-subfamily kinases ZC1(SEQ ID NO:13), ZC2 (SEQ ID NO:14), and ZC4 (SEQ ID NO:16), sharing62.0%, 61.0%, and 42.5% amino acid identity, respectively and shares46.7% amino acid identity to a C. elegans kinase encoded by the cosmidZC504.4 (GB:Z50029).

The 255 amino acid catalytic domain of human ZC3 is most related tohuman STE20-subfamily kinases, ZC1 (SEQ ID NO:13), ZC2 (SEQ ID NO:14),SOK-1 (GB:X99325), KHS2 (SEQ ID NO:18), GCK (GB:U07349), SULU1 (SEQ IDNO:22), and GEK2 (SEQ ID NO:107), and to the C. elegans kinase encodedby the cosmid ZC504.4 (GB:Z50029) sharing 90.6%, 89.3%, 49.0%, 48.3%,45.0%, 43.1%, 42.3% and 76.7% amino acid identity, respectively. ZC1contains the potential “TPY” regulatory phosphorylation site in itsactivation loop. This “TPY” motif is conserved in other STE20-relatedkinases, including ZC1, ZC2, GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.

Immediately C-terminal to the kinase domain of human ZC3 is a 221 aminoacid region predicted to form a coiled-coil structure based on the Lupasalgorithm (supra). This region of ZC3 is most homologous to humanSTE20-subfamily kinases, ZC1 (SEQ ID NO:13), ZC2 (SEQ ID NO:14), andGEK2 (SEQ ID NO:107), sharing 66.9%, 61.5%, and 27.5% identity, as wellas to rat PLC-beta (GB:A45493) and human PITSLRE (GB:H54024) sharing29.6% and 25.9% amino acid identity, respectively. The C. eleganshomologue ZC504.4 shares 26.8% sequence identity over this region.

Within the predicted coiled-coil domain of human ZC3, and the relatedZC1, is a region predicted to form a leucine zipper(Leu-X6-Leu-X6-Leu-X6-Leu-X20-Leu-X6-Leu) (SEQ ID NO: 149). The factthat this leucine repeat exists within a predicted coiled-coil structuresuggests that the leucine zipper may have a high probability of servingas a dimerization interface (Hirst, J. D. et al Protein Engineering 9657-662 (1996)) mediating a potential inter- or intra-moleculardimerization of human ZC3.

The 204 amino acid proline-rich region of human ZC3 is most related tohuman STE20-subfamily kinases, ZC1 (SEQ ID NO:13) and ZC2 (SEQ IDNO:14), sharing 66.9% and 61.5% amino acid identity, respectively.

ZC3 contains two of the potential “PxxP” (SEQ ID NO: 148) SH3domain-binding motifs (II and III) found within the proline-rich regionof human ZC1. Motif II is conserved in ZC1, ZC2, ZC4 and C. elegansZC504.4; Motif II is conserved in ZC1, ZC2 and ZC4. Motifs II and III ofmurine NIK have been shown to bind the SH3 motif of the adaptor moleculeNck. From this evidence, human ZC3 may have the potential to bind to Nckor other SH3 or WW domain-containing proteins and participate in growthfactor-induced signaling pathways.

The 303 amino acid acid spacer region of human ZC3 is most related tohuman STE20-subfamily kinases, ZC1 (SEQ ID NO:13) and ZC2 (SEQ ID NO:14)sharing 30.1%, and 27.1% amino acid identity, respectively. The C.elegans homologue ZC504.4 lacks nearly the entire spacer region of ZC3.

The 343 amino acid C-terminal of human ZC3 is most related to humanSTE20-subfamily kinases, ZC1 (SEQ ID NO:13), ZC2 (SEQ ID NO:14) and ZC4(SEQ ID NO:16), sharing 89.2%, 88.9%, and 42.5%, amino acid identity,respectively. The C. elegans homologue ZC504.4 also shares 67.2% aminoacid identity with the C-tail of human ZC3. A lower, yet significant,homology is also evident to human GCK (GB:U07349), as well as to thenon-kinases murine citron (GB:U07349) and the S. cerevisiae ROM2 protein(GB:U19103), a Rho1 GDP/GTP exchange factor, with 21.6%, 32.4% and 22.9%amino acid identity, respectively.

The sequence similarity of the C-terminal region of ZC3 to proteins thathave potential Rab- or Rho-binding domains suggests that ZC3, like ZC1and ZC2, may signal through a small G-protein-dependant pathway.

Mammalian ZC4

The 3,684 bp human ZC4 nucleotide sequence of the complete cDNA encodesa polypeptide of 1,227 amino acids (SEQ ID NO:105) with a predictedmolecular mass of 138,205 Daltons. Analysis of the deduced amino acidsequence predicts ZC4 to be an intracellular STE20-subfamily kinase,lacking both a signal sequence and a transmembrane domain. Thefull-length ZC4 protein contains a 25 amino acid N-terminus, a 265 aminoacid catalytic domain with all the motifs characteristic of aserine/threonine kinase, a 108 amino acid region predicted to form acoiled-coil structure, a 231 amino acid proline-rich region, a 40 aminoacid region predicted to form a coiled-coil structure spacer region, a204 amino acid spacer region (domain B), followed by a 355 amino acidC-terminal domain containing a potential Rab/Rho-binding region (domainC).

ZC4 is most closely related to human ZC1 (SEQ ID NO:13, also known ashuman HGK, human KIAA0687, murine NIK, human AC005035, human NIK, and C.elegans MIG-15), ZC2 (SEQ ID NO:14, similar to partial sequence humanKIAA0551), and ZC3 (SEQ ID NO:15). An assembled genomic fragment in thedatabase (Z83850) is identical to ZC4, except for inappropriateidentification of the exon boundaries. (Abo et al. (1998) EMBO J. 17:6527-6540.)

The 25 amino acid N-terminal domain of human ZC4 shares weak homology tohuman ZC1 in its C-terminal extent, but otherwise does not reveal anysignificant homologies to the protein database.

The 265 amino acid catalytic domain of human ZC4 is most related tohuman ZC1 (SEQ ID NO:13), ZC3 (SEQ ID NO:15), and ZC2 (SEQ ID NO:14),sharing 63%, 64% and 62% amino acid identity, respectively.

Immediately C-terminal to the kinase domain of human ZC4 is a 108 aminoacid region predicted to form a coiled-coil structure based on the Lupasalgorithm (supra). This region is most related to human ZC1 (SEQ IDNO:13), ZC3 (SEQ ID NO:15), and ZC2 (SEQ ID NO:14), sharing 29%, 25% and20% amino acid identity, respectively.

The 231 amino acid proline-rich region of human ZC4 does not reveal anysignificant homologies to the protein database. This region of ZC4contains two “PxxP” (SEQ ID NO: 148) motifs that could potentially bindto proteins containing SH3 or WW domains and allow ZC4 to participate ingrowth factor activated signaling pathways. In addition, within thepro-rich domain of human ZC4 is a region predicted to form a leucinezipper (Leu-X6-Leu-X6-Leu-X6-Leu-X20-Leu-X6-Leu) (SEQ ID NO: 149) whichmay serve as a dimerization interface. The ZC STE20 subfamily kinases(ZC1, ZC2 and ZC3) have similarly located “PxxP’ (SEQ ID NO: 148) motifsand potential Leu zippers.

Immediately C-terminal to the proline-rich region of human ZC4 is a 40amino acid region also predicted to form a coiled-coil structure basedon the Lupas algorithm. This region of human ZC4 does not reveal anysignificant homologies to the protein database.

The 204 amino acid acidic- and serine-rich domain “B” of ZC4 does notreveal any significant homologies to the protein database.

The 355 amino acid C-terminal of human ZC4 is most related to human ZC1(SEQ ID NO:13), ZC3 (SEQ ID NO:15), and ZC2 (SEQ ID NO:14), sharing 43%,42% and 42% amino acid identity, respectively.

The sequence similarity of the C-terminal region of ZC4 to proteins thathave potential Rab- or Rho-binding domains suggests that ZC4, like otherZC-subfamily STE20 kinases, may signal through a smallG-protein-dependant pathway.

Mammalian KHS2

The 4023 bp human KHS2 nucleotide sequence encodes a polypeptide of 894amino acids (SEQ ID NO:18) with a predicted molecular mass of 101,327daltons. Analysis of the deduced amino acid sequence predicts KHS2 to bean intracellular serine/threonine kinase, lacking both a signal sequenceand transmembrane domain. The full-length KHS2 protein contains a 13amino acid N-terminus, a 260 amino acid catalytic domain with all themotifs characteristic of a serine/threonine kinase, a 73 amino acidspacer region, a 188 proline-rich region, followed by a 360 amino acidC-terminal domain containing a potential Rab/Rho-binding site.

KHS2 is most closely related to the human STE20-subfamily kinases KHS1(GB:U177129), GCK (GB:U07349), and HPK1 (GB:U07349), sharing 65.5%,51.9%, and 44.9% amino acid identity, respectively and shares 38.5%amino acid identity to a C. elegans STK (GB:U55363).

The 13 amino acid N-terminal domain of human KHS2 does not reveal anysignificant homologies that might suggest a potential function for thisdomain when examined by a Smith-Waterman alignment to the nonredundantprotein database. Human KHS2 lacks a glycine residue at position 2, andis therefore unlikely to undergo myristylation.

The 260 amino acid catalytic domain of human KHS2 is most related tohuman STE20-subfamily kinases KHS1 (GB:U177129), GCK (GB:U07349), HPK1(GB:U66464), SOK-1 (GB:X99325), MST1 (GB:U18297), ZC1 (SEQ ID NO:13),and to the C. elegans kinase (GB:U55363), sharing 85.4%, 75.1%, 67.7%,51.4%, 48.1%, 49.8% and 72.0% amino acid identity, respectively. KHS2contains the potential “TPY” regulatory phosphorylation site in itsactivation loop. This “TPY” motif is conserved in other STE20-relatedkinases, including ZC1, ZC2, ZC3, ZC4, GEK2, SULU1, SULU3, PAK4 andPAK5.

The 73 amino acid acid spacer region of human KHS2 is most related tohuman STE20-subfamily kinases, KHS1 (GB:U177129), HPK1 (GB:U66464) andGCK (GB:U07349), sharing 60.3%, 43.5% and 44.0%, amino acid identity,respectively.

The 188 amino acid proline-rich region of human KHS2 is most related tohuman STE20-subfamily kinases, HPK1 (GB:U66464), GCK (GB:U07349) andKHS1 (GB:U177129), sharing 33.3%, 31.9% and 31.4%, amino acid identity,respectively.

Two potential “PxxP” (SEQ ID NO: 148) SH3 domain-binding motifs (I andII) are found within the proline-rich region of human KHS2. Motif I isconserved with human KHS1 and HPK1; motif II is conserved with GCK andKHS2. A 192 amino acid region of human HPK1 containing motif II has beenshown to bind to the C-terminal SH3 motif of the adaptor molecule Grb2(Anafi, M et al, J. Biol. Chem. J. 272, 27804-27811 (1997)). Human KHS2may bind SH3 or WW domain-containing proteins through this proline-richregion.

The 360 amino acid C-terminal of human KHS2 is most related to KHS1(GB:U177129), GCK (GB:U07349) and HPK1 (GB:U66464), and to the C.elegans kinase (GB:U55363), sharing 74.9%, 54.8%, 42.9%, and 31.0%,amino acid identity, respectively. GCK is a STE20-family kinase whoseC-terminal domain has been shown to bind the small G-protein Rab8 (Ren,M. et al., Proc. Natl. Acad. Sci. 93, 5151-5155 (1996)).

Mammalian SULU1

The 4196 bp human SULU1 nucleotide sequence encodes a polypeptide of 898amino acids (SEQ ID NO:22) with a predicted molecular mass of 105,402daltons. Analysis of the deduced amino acid sequence predicts SULU1 tobe an intracellular serine/threonine kinase, lacking both a signalsequence and transmembrane domain. The full-length SULU1 proteincontains a 21 amino acid N-terminus, a 256 amino acid catalytic domainwith all the motifs characteristic of a serine/threonine kinase, a 150amino acid spacer region, a 210 amino acid region predicted to form acoiled-coil structure, a 114 amino acid spacer region and a 147 aminoacid C-terminal domain predicted to form a coiled-coil structure.

SULU1 is most closely related to the STE20-subfamily kinases murineSULU3 (SEQ ID NO:24), human SULU3 (SEQ ID NO:23), and to the C. eleganskinase SULU (GB:U11280), sharing 68.9%, 72.2% and 38.2% amino acididentity, respectively.

The 21 amino acid N-terminal domain of human SULU1 is most related tomurine SULU3 (SEQ ID NO:24) and to the C. elegans kinase SULU(GB:U11280), sharing 86.3% and 62.3% amino acid identity. Human SULU1lacks a glycine residue at position 2, and is therefore unlikely toundergo myristoylation. A Smith-Waterman search of the nonredundantprotein database does not reveal any significant homologies that mightsuggest a potential function for this domain.

The 256 amino acid catalytic domain of human SULU1 is most related tomurine SULU3 (SEQ ID NO:24), and to human SOK-1 (GB:X99325), STLK2 (SEQID NO:5), MST1 (GB:U18297), PAK1 (GB:U24152), ZC2 (SEQ ID NO:14), andKHS2 (SEQ ID NO:18) sharing 86.3%, 48.1%, 46.9%, 45.2%, 43.3%, 43.1% and42.0% amino acid identity, respectively. The C. elegans SULU STK(GB:U11280) shares 62.3% sequence identity over this region. SULU1contains the potential “TPY” regulatory phosphorylation site in itsactivation loop. This “TPY” motif is conserved in other STE20-relatedkinases, including ZC1, ZC2, ZC3, ZC4, GEK2, KHS2, SULU3, PAK4 and PAK5.

The 150 amino acid spacer region of human SULU1 is most related to humanSULU3 (SEQ ID NO:23) and to the C. elegans kinase (GB:U11280), sharing53.5% and 10.4% amino acid identity, respectively.

Immediately C-terminal to the spacer region of human SULU1 is a 210amino acid region predicted to form a coiled-coil structure based on theLupas algorithm. This region of SULU1 is most related to SULU3 (SEQ IDNO:23), the C. elegans SULU kinase (GB:U11280), GEK 2 (SEQ ID NO:107)and ZC1 (SEQ ID NO:13), sharing 68.6%, 26.8%, 23.2%, and 22.8% aminoacid identity, respectively.

The 114 amino acid spacer region human SULU1 is most related to humanSULU3 (SEQ ID NO:24) with 73.7% amino acid sequence identity. A lower,yet significant, homology is also evident to murine PITSLRE (GB:U04824)and DLK (GB:A55318), human ZC1 (SEQ ID NO:13) and GEK 2 (SEQ ID NO:107),as well as to the C. elegans SULU STK (GB:U11280), sharing 39.7%, 35.4%,29.5%, 23.6% and 37.6% amino acid identity, respectively.

Immediately C-terminal to the second spacer region of human SULU1 is a147 amino acid region predicted to form a coiled-coil structure based onthe Lupas algorithm. This region of SULU1 is most related to human SULU3(SEQ ID NO:24), ZC1 (SEQ ID NO:13) and GEK 2 (SEQ ID NO:107), as well asto the C. elegans SULU STK (GB:U11280), sharing 73.3%, 28.4%, 26.1% and39.5%, amino acid identity, respectively.

Mammalian (Human) SULU3

The 3824 bp partial cDNA human SULU3 nucleotide sequence encodes apolypeptide of 786 amino acids (SEQ ID NO:23) with a predicted molecularmass of 92,037 daltons. Analysis of the deduced amino acid sequencepredicts SULU3 to be an intracellular serine/threonine kinase lacking atransmembrane domain. The N-terminally truncated human SULU3 proteincontains a 66 amino acid partial catalytic domain followed by a 149amino acid spacer region, a 210 amino acid region predicted to form acoiled-coil structure, a second spacer region of 114 amino acids, a 247amino acid C-terminal region predicted to form a second coiled-coilstructure and a 100 amino acid C-terminal tail.

Human SULU3 is most closely related murine SULU3 (SEQ ID NO:24), humanSULU1 (SEQ ID NO:22), and to the C. elegans SULU kinase (GB:U11280),sharing 66.3%, 68.9% and 32.9% amino acid identity, respectively. Thehigh sequence homology between murine and human SULU3 suggests thatthese two proteins are orthologs of each other.

The 66 amino acid partial catalytic domain of human SULU3 is mostrelated to murine SULU3 (SEQ ID NO:24), and to the human STE20 subfamilykinases ZC1 (SEQ ID NO:13), STE20 (GB:X99325), KHS1(GB:U177129) and GEK2 (SEQ ID NO:107), as well as to the C. elegans SULU kinase (GB:U11280),sharing 83.3%, 47.0%, 45.5%, 43.5%, 41.8% and 55.6% amino acid identity,respectively.

The 149 amino acid spacer region of human SULU3 is most related tomurine SULU3 (SEQ ID NO:24), human STE20 (GB:X99325), MST1 (GB:U18297),and to the C. elegans SULU kinase (GB:U11280) sharing 98.7%, 21.9% and21.8% amino acid identity, respectively.

Immediately C-terminal to the first spacer region of human SULU3 is a210 amino acid region predicted to form a coiled-coil structure based onthe Lupas algorithm. This region of SULU3 is most related to murineSULU3 (SEQ ID NO:24), and to human SULU1 (SEQ ID NO:22), ZC1 (SEQ IDNO:13) and GEK 2 (SEQ ID NO:107), as well as to the C. elegans SULUkinase (GB:U11280), sharing 99.5%, 68.6%, 27.4% and 22.5% amino acididentity, respectively.

The 114 amino acid second spacer region of human SULU3 is most relatedto murine SULU3 (SEQ ID NO:24), and to human SULU1 (SEQ ID NO:22) GEK 2(SEQ ID NO:107), and ZC1 (SEQ ID NO:13), as well as to the C. elegansSULU kinase (GB:U11280), sharing 99.1%, 73.7%, 24.6%, 24.1% and 41.2%amino acid identity, respectively.

Immediately C-terminal to the second spacer region of human SULU3 is a247 amino acid region predicted to form a coiled-coil structure based onthe Lupas algorithm (supra). This region of SULU3 is most related tohuman SULU1 (SEQ ID NO:22) and ZC1 (SEQ ID NO:13) as well as to ratPKN-(GB:D26180) murine p160 ROCK1 (GB:U58512), and the C. elegans SULUkinase (GB:U11280), sharing 73.7%, 26.7%, 24.0% and 21.0% amino acididentity, respectively.

The 100 amino acid C-tail of human SULU3 is most related to a humanprion protein (GB:L38993), with 45.0% amino acid identity.

Mammalian (Murine) SULU3

The 2249 bp murine, partial cDNA SULU3 nucleotide sequence encodes apolypeptide of 748 amino acids (SEQ ID NO:24) with a predicted molecularmass of 87,520 daltons. Analysis of the deduced amino acid sequencepredicts SULU3 to be an intracellular serine/threonine kinase, lackingboth a signal sequence and transmembrane domain. The partial murineSULU3 protein contains a 25 amino acid N-terminus, a 248 amino acidcatalytic domain with all the motifs characteristic of aserine/threonine kinase, a 149 amino acid spacer region, a 210 aminoacid region predicted to form a coiled-coil structure, and a 116 aminoacid spacer region.

Murine SULU3 is most closely related to human SULU3 (SEQ ID NO:23) andSULU1 (SEQ ID NO:22), as well as to the C. elegans SULU kinase (GB:U11280), sharing 97.0%, 72.3% and 38.4% amino acid identity, respectively.The high sequence homology between murine and human SULU3 suggests thatthese two proteins are orthologs.

The 25 amino acid N-terminal domain of murine SULU3 is most related tohuman SULU1 (SEQ ID NO:22) and to the C. elegans SULU kinase(GB:U11280), sharing 70.0% and 44.4% amino acid identity, respectively.

Murine SULU3 lacks a glycine residue at position 2, and is thereforeunlikely to undergo myristoylation. A Smith-Waterman search of thenonredundant protein database does not reveal any significant homologiesthat might suggest a potential function for this domain.

The 248 amino acid catalytic domain of murine SULU3 is most related tohuman SULU1 (SEQ ID NO:22), STE20 (GB:X99325), ZC1 (SEQ ID NO:13), andKHS1 (GB:U77129), as well as to the C. elegans SULU kinase (GB:U11280),sharing 86.7%, 46.6%, 43.3%, 59.4% amino acid identity, respectively.Murine SULU3 contains the potential “TPY” regulatory phosphorylationsite in its activation loop. This “TPY” motif is conserved in otherSTE20-related kinases, including ZC2, ZC3, ZC4, GEK2, KHS2, SULU1,SULU3, PAK4 and PAK5.

The 149 amino acid spacer of murine SULU3 is most related to human SULU3(SEQ ID NO:23), SULU1 (SEQ ID NO:22), and STE20 (GB:X99325), as well asto the C. elegans SULU (GB:U11280) and the S. cerevisiae STE20(GB:L04655) kinases, sharing 98.7%, 53.4%, 21.9%, 59.4% and 21.9% aminoacid identity, respectively.

Immediately C-terminal to the spacer region of murine SULU3 is a 210amino acid region predicted to form a coiled-coil structure based on theLupas algorithm. This region of murine SULU3 is most related to humanSULU3 (SEQ ID NO:23), ZC1 (SEQ ID NO:13), and GEK 2 (SEQ ID NO:107), aswell as to the C. elegans SULU kinase (GB:U11280), sharing 99.5%, 27.4%,22.5% and 29.2% amino acid identity, respectively.

The 116 amino acid C-terminal spacer region of murine SULU3 is mostrelated to human SULU3 (SEQ ID NO:23), GEK 2 (SEQ ID NO:107), and ZC1(SEQ ID NO:13), well as to the C. elegans SULU kinase (GB:U11280),sharing 98.3%, 24.6%, 24.1% and 40.5% amino acid identity, respectively.

Mammalian (Murine/Human) SULU3

The 2249 bp murine SULU3 and the 3824 bp human SULU3 cDNAs contain a1620 nucleotide overlap (541 amino acids) with 90% and 98% DNA and aminoacid sequence identity, respectively. Owing to the high degree ofsequence identity in this extended overlap, we propose that these arefunctional orthologues of a single gene. The combined murine/human 4492bp SULU3 sequence encodes a polypeptide of 1001 amino acids (SEQ IDNO:31) with a predicted molecular mass of 116,069 daltons. Analysis ofthe deduced amino acid sequence predicts SULU3 to be an intracellularserine/threonine kinase, lacking both a signal sequence andtransmembrane domain. SULU3 contains a 25 amino acid N-terminus, a 248amino acid catalytic domain with all the motifs characteristic of aserine/threonine kinase, a 149 amino acid spacer region, a 210 aminoacid region predicted to form a coiled-coil structure and a secondspacer region of 114 amino acids, a 247 amino acid C-terminal regionpredicted to form a second coiled-coil structure and a 100 amino acidC-terminal tail. The murine SULU3 clone lacks the region from the secondC-terminal coiled-coil to the C-terminus, whereas the human clone lacksthe N-terminal domain, and all but 66 amino acids of the 248 amino acidkinase domain.

SULU3 is most closely related to SULU1 (SEQ ID NO:22) and the C. elegansSULU kinase (GB:U11280) sharing 72.3% and 38.4% amino acid identity,respectively.

The 25 amino acid N-terminal domain of SULU3 is most related to humanSULU1 (SEQ ID NO:22) and to the C. elegans SULU kinase (GB:U11280),sharing 70.0% and 44.4% amino acid identity, respectively. SULU3 lacks aglycine residue at position 2, and is therefore unlikely to undergomyristylation. A Smith-Waterman search of the nonredundant proteindatabase does not reveal any significant homologies that might suggest apotential function for this domain.

The 248 amino acid catalytic domain of SULU3 is most related to humanSULU1 (SEQ ID NO:22), SOK-1 (GB:X99325), ZC1 (SEQ ID NO:13), KHS1(GB:U77129) and the C. elegans SULU kinase (GB:U11280), sharing 86.7%,46.6%, 43.3%, 42.0% and 59.4% amino acid identity, respectively. SULU3contains the potential “TPY” regulatory phosphorylation site in itsactivation loop. This “TPY” motif is conserved in other STE20-relatedkinases, including ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, PAK4 and PAK5.

The 149 amino acid spacer of SULU3 is most related to SULU1 (SEQ IDNO:22) and SOK-1 (GB:X99325), and to the C. elegans SULU (GB:U11280),and S. cerevisiae STE20 (GB:L04655) kinases, sharing 53.4%, 21.9%, 59.4%and 21.9% amino acid identity, respectively.

Immediately C-terminal to the spacer region of SULU3 is a 210 amino acidregion predicted to form a coiled-coil structure based on the Lupasalgorithm. This region is most related to ZC1 (SEQ ID NO:13), GEK 2 (SEQID NO:107), and the C. elegans SULU kinase (GB:U11280), sharing 27.4%22.5% and 29.2% amino acid identity, respectively.

The 114 amino acid spacer region of SULU3 is most related to human SULU1(SEQ ID NO:22), GEK 2 (SEQ ID NO:107), ZC1 (SEQ ID NO:13), and to the C.elegans SULU kinase (GB:U11280), sharing 73.7%, 24.6%, 24.1% and 41.2%amino acid identity, respectively.

Immediately C-terminal to the second spacer region of SULU3 is a 247amino acid region predicted to form a coiled-coil structure based on theLupas algorithm. This region of SULU3 is most related to human SULU1(SEQ ID NO:22) and ZC1 (SEQ ID NO:13), as well as to rat PKN(GB:D26180), murine p160 ROCK1 (GB:U58512) and the C. elegans SULUkinase (GB:U11280), sharing 73.7%, 26.7%, 24.0%, 21.0% and 37.6% aminoacid identity, respectively.

The 100 amino acid C-tail of SULU3 is most related to a human prionprotein (GB:L38993) with 45.0% amino acid identity.

Mammalian GEK2

The 2926 bp human GEK2 nucleotide sequence of the complete cDNA encodesa polypeptide of 968 amino acids (SEQ ID NO:107) with a predictedmolecular mass of 112,120 daltons. Analysis of the deduced amino acidsequence predicts GEK2 to be an intracellular serine/threonine kinase,lacking both a signal sequence and transmembrane domain. The completeGEK2 protein contains a 33 amino acid N-terminus, a 261 amino acidcatalytic domain with all the motifs characteristic of aserine/threonine kinase, a 43 amino acid spacer region, a 135 amino acidproline-rich region, a 252 amino acid region predicted to form acoiled-coil structure followed by a 244 amino acid region also predictedto form a coiled-coil structure.

GEK2 is most closely related to rat AT1-46 (GB:U33472) (a partial cDNAthat extends from the middle of the first potential coiled-coil domainof GEK2 to the C-terminus), murine LOK (GB:D89728), Xenopus laevispolo-like kinase 1 (GB:AF100165), and human SLK (GB:AB002804), sharing91.3%, 88.5%, 65.0%, and 44.7% amino acid identity, respectively. Thehigh sequence homology between human GEK2, murine LOK and rat AT1-46suggests that human GEK2 is a highly related protein to the rodentforms, or alternatively, its orthologue. Recently, a full-length versionof GEK2 was reported (STK10 or human LOK AB015718). The 968 amino acidsequence is 99% identical to GEK2 (SEQ ID NO:107).

The 33 amino acid N-terminal domain of human GEK2 is most related tomurine LOK (GB:D89728) and to human SLK (GB:AB002804), sharing 100% and54.5% amino acid identity, respectively.

Human GEK2 lacks a glycine residue at position 2, and is thereforeunlikely to undergo myristylation. A Smith-Waterman search of thenonredundant protein database does not reveal any significant homologiesthat might suggest a potential function for this domain.

The 261 amino acid catalytic domain of human GEK2 is most related tomurine LOK (GB:D89728), rat AT 1-46 (GB:D89728) and human SLK(GB:AB002804) as well as to a C. elegans kinase (GB:Z81460), sharing97.7%, 90.8%, 54.5% and 55.9% amino acid identity, respectively. GEK2contains the potential “TPY” regulatory phosphorylation site in itsactivation loop. This “TPY” motif is conserved in other STE20-relatedkinases, including ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3, PAK4 andPAK5.

The 43 amino acid spacer region of human GEK2 is most related to murineLOK (GB:D89728) and to human SLK, sharing 83.7% and 77.6% amino acididentity, respectively.

The 135 amino acid proline-rich region of human GEK2 is most related tomurine LOK (GB:D89728) with 66.2% amino acid identity, respectively.Within the proline-rich region of human GEK2 is a potential “PxxP” (SEQID NO: 148) SH3-binding domain conserved with murine LOK.

Immediately C-terminal to the proline-rich region of human GEK2 is a 252amino acid region predicted to form a coiled-coil structure based on theLupas algorithm. This region of human GEK2 is most related to rat AT1-46(GB:D89728), murine LOK (GB:D89728) and human SLK (GB:AB002804), and ZC2(SEQ ID NO:14), sharing 90.8%, 86.9%, 42.2%, and 29.7% amino acididentity, respectively.

Immediately C-terminal to the predicted coiled-coil structure of humanGEK2 is a second potential coiled-coil structure of 244 amino acidspredicted based on the Lupas algorithm. This region of human GEK2 ismost related to rat AT1-46 (GB:D89728) and murine LOK (GB:D89728) aswell as to human SLK (GB:AB002804) and ZC1 (SEQ ID NO:13), sharing91.8%, 92.6%, 70.4% and 26.7% amino acid identity, respectively. The C.elegans kinase (GB:Z81460) shares 31.5% amino acid sequence identityover this region.

Mammalian PAK4

The 3604 bp human PAK4 nucleotide sequence encodes a polypeptide of 681amino acids (SEQ ID NO:29) with a predicted molecular mass of 74,875daltons. Analysis of the deduced amino acid sequence predicts PAK4 to bean intracellular serine/threonine kinase, lacking both a signal sequenceand transmembrane domain. The full-length PAK4 protein contains a 51amino acid N-terminus predicted to contain a rac-binding motif, a 173amino acid insert relative to the known mammalian PAK proteins, a 169amino acid spacer region, a 265 amino acid catalytic domain with all themotifs characteristic of a serine/threonine kinase and a 23 amino acidC-terminal tail.

PAK4 is most closely related to human PAK5 (SEQ ID NO:30), PAK1 (GB:U24152), and PAK65 (GB:U25975), as well as to a C. elegans kinase (GB:Z74029), sharing 76.8%, 49.5%, 49.8%, and 34.6% amino acid identity,respectively.

The 51 amino acid N-terminal domain of human PAK4 is most related tohuman PAK1 (GB:U24152), and PAK65 (GB:U25975), as well as to a C.elegans kinase (GB: Z74029), sharing 50.0%, 50.0% and 49.0% amino acididentity, respectively.

The 10 amino acid region at positions 13-23 of human PAK4 fits theconsensus for a Cdc42/Rac-binding motif (SXPX4-6HXXH) (SEQ ID NO: 150)(Burbelo, P. D., Dreschel, D. and Hall, A. J. Bio. Chem. 270,29071-29074 (1995)).

The 173 amino acid insert of human PAK4, relative to the known mammalianPAK proteins, is most related to a C. elegans kinase (GB: Z74029) with39.0% amino acid identity. A Smith-Waterman search of the nonredundantprotein database does not reveal any significant homologies that mightsuggest a potential function for this region.

The 169 amino acid spacer of human PAK4 does not reveal any significanthomologies that might suggest a potential function for this region.

The equivalent spacer region in PAK1 binds to the guanine nucleotideexchange factor PIX (Manser, E. et al (1998) Molecular Cell, 1,183-192). Since PAK4 differs substantially from PAK1 over this region,the spacer domain of PAK4 may differ in its guanine nucleotide exchangefactor binding specificity, relative to PAK1.

The 265 amino acid catalytic domain of human PAK4 is most related tohuman PAK5 (SEQ ID NO:30), PAK1 (GB:U24152), GCK (GB:U07349), SOK-1(GB:X99325), and SLK (GB:AB002804), as well as to the C. elegans (GB:Z74029), and S. cerevisiae STE20-related kinases (GB:L04655), sharing95.9%, 51.7%, 41.3%, 39.8%, 37.4%, 60.2% and 47.9% amino acid identity,respectively. PAK4 contains the potential “TPY” regulatoryphosphorylation site in its activation loop. This “TPY” motif isconserved in other STE20-related kinases, including ZC1, ZC2, ZC3, ZC4,GEK2, KHS2, SULU1, SULU3 and PAK5.

The 23 amino acid C-tail of human PAK4 contains a sequence that ishomologous to a G-protein beta subunit binding site (Leeuw, T. et al.Nature, 391, 191-195 (1998)). PAK4 has, therefore, the potential to beactivated by both Cdc42- as well as G-protein-dependant pathways.

Mammalian PAK5

The 2,806 bp human PAK5 nucleotide sequence of the complete cDNA encodesa polypeptide of 591 amino acids (SEQ ID NO:103) with a predictedmolecular mass of 64,071 Daltons. Analysis of the deduced amino acidsequence predicts PAK5 to be an intracellular STE20-subfamily kinase,lacking both a signal sequence and transmembrane domain. The full-lengthPAK5 protein contains a 52 amino acid N-terminus predicted to contain ap21 (small G-protein) binding domain (PDB or CRIB), a 121 amino acidinsert relative to the known mammalian PAK proteins, a 134 amino spacerregion, a 265 amino acid catalytic domain with all the motifscharacteristic of a serine/threonine kinase and a 19 amino acidC-terminal tail.

PAK5 is most closely related to Human PAK4 (SEQ ID NO:29), Drosophilamelanogaster PAK (also known as “mushroom bodies tiny”) (AJ011578),C45B11.1b from C. elegans (Z74029), and human PAK3 (Q13177) sharing 48%(327/674 aa), 50% (330/651 aa), 43% (234/435 aa excluding gap), and 47%(190/405 aa excluding gap) amino acid identity, respectively. Recently,a full length version of PAK5 was reported (PAK4 AF005046) whose 591amino acid sequence is identical to PAK5 (SEQ ID NO:103). (Abo, et al.(1998) EMBO J. 17: 6527-6540).

The 52 amino acid N-terminal domain of human PAK5 is most related tohuman PAK4 (SEQ ID NO:29), Drosophila melanogaster PAK (AJ011578),C45B11.1b from C. elegans (Z74029), and human PAK3 (Q13177), sharing65%, 57%, 54%, and 53% amino acid identity, respectively.

The 10 amino acid region at positions 12-22 of human PAK5 (FIG. 18) fitsthe consensus for a small G-protein binding domain (PDB or CRIB)(SXPX4-6HXXH) (SEQ ID NO: 150) (Burbelo, P. D., Dreschel, D. and Hall,A. J. Bio. Chem. 270, 29071-29074 (1995), hereby incorporated byreference herein in its entirety including any figures, tables, ordrawings.).

The 121 amino acid insert of human PAK5 shares 43% amino acid identitywith a similar domain from PAK4 (SEQ ID NO:29), but that is absent fromother known PAKs.

The equivalent spacer region in PAK1 binds to the guanine nucleotideexchange factor PIX (Manser, E. et al (1998) Molecular Cell, 1, 183-192hereby incorporated by reference herein in its entirety including anydrawings, figures, or tables.). Since PAK5 differs substantially fromPAK1 over this region, the spacer domain of PAK5 may differ in itsguanine nucleotide exchange factor binding specificity, relative toPAK1.

The 134 amino acid collagen-like region of human PAK5 shares 34% aminoacid identity to pro-α I type collagen from several species and is notpresent in other known PAKs.

The 265 amino acid catalytic domain of human PAK5 is most related tohuman PAK4 (SEQ ID NO:29), Drosophila melanogaster PAK (AJ011578),C45B11.1b from C. elegans (Z74029), and human PAK3 (Q13177), sharing78%, 80%, 61%, and 55% amino acid identity, respectively. PAK5 alsocontains the potential “TPY” regulatory phosphorylation site in itsactivation loop. This “TPY” motif is conserved in other STE20-relatedkinases, including ZC1, ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3 andPAK4.

The 19 amino acid C-tail shares 80% amino acid identity to a PAK-likehomologue identified from genomic DNA (AL031652). Furthermore, thisC-terminal region of human PAK5 contains a sequence that is homologousto a G-protein beta subunit binding site (Leeuw, T. et al. Nature, 391,191-195 (1998) hereby incorporated by reference herein in its entiretyincluding any figures, tables, or drawings). PAK5 has, therefore, thepotential to be activated by both, Cdc42 as well as G-protein-dependantpathways.

V. Antibodies, Hebridomas, Methods of Use and Kits for Detection ofSTE20-Related Kinases

The present invention relates to an antibody having binding affinity toa kinase of the invention. The polypeptide may have the amino acidsequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or a functionalderivative thereof, or at least 9 contiguous amino acids thereof(preferably, at least 20, 30, 35, or 40 or more contiguous amino acidsthereof).

The present invention also relates to an antibody having specificbinding affinity to a kinase of the invention. Such an antibody may beisolated by comparing its binding affinity to a kinase of the inventionwith its binding affinity to other polypeptides. Those which bindselectively to a kinase of the invention would be chosen for use inmethods requiring a distinction between a kinase of the invention andother polypeptides. Such methods could include, but should not belimited to, the analysis of altered kinase expression in tissuecontaining other polypeptides.

The STE20-Related kinases of the present invention can be used in avariety of procedures and methods, such as for the generation ofantibodies, for use in identifying pharmaceutical compositions, and forstudying DNA/protein interaction.

The kinases of the present invention can be used to produce antibodiesor hybridomas. One skilled in the art will recognize that if an antibodyis desired, such a peptide could be generated as described herein andused as an immunogen. The antibodies of the present invention includemonoclonal and polyclonal antibodies, as well fragments of theseantibodies, and humanized forms. Humanized forms of the antibodies ofthe present invention may be generated using one of the procedures knownin the art such as chimerization or CDR grafting.

The present invention also relates to a hybridoma which produces theabove-described monoclonal antibody, or binding fragment thereof. Ahybridoma is an immortalized cell line which is capable of secreting aspecific monoclonal antibody.

In general, techniques for preparing monoclonal antibodies andhybridomas are well known in the art (Campbell, “Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology,” Elsevier Science Publishers, Amsterdam, The Netherlands, 1984;St. Groth et al., J. Immunol. Methods 35: 1-21, 1980). Any animal(mouse, rabbit, and the like) which is known to produce antibodies canbe immunized with the selected polypeptide. Methods for immunization arewell known in the art. Such methods include subcutaneous orintraperitoneal injection of the polypeptide. One skilled in the artwill recognize that the amount of polypeptide used for immunization willvary based on the animal which is immunized, the antigenicity of thepolypeptide and the site of injection.

The polypeptide may be modified or administered in an adjuvant in orderto increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells. Any oneof a number of methods well known in the art can be used to identify thehybridoma cell which produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, western blot analysis, or radioimmunoassay (Lutz et al., Exp.Cell Res. 175: 109-124, 1988). Hybridomas secreting the desiredantibodies are cloned and the class and subclass are determined usingprocedures known in the art (Campbell, “Monoclonal Antibody Technology:Laboratory Techniques in Biochemistry and Molecular Biology”, supra,1984).

For polyclonal antibodies, antibody-containing antisera is isolated fromthe immunized animal and is screened for the presence of antibodies withthe desired specificity using one of the above-described procedures. Theabove-described antibodies may be detectably labeled. Antibodies can bedetectably labeled through the use of radioisotopes, affinity labels(such as biotin, avidin, and the like), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, and the like) fluorescentlabels (such as FITC or rhodamine, and the like), paramagnetic atoms,and the like. Procedures for accomplishing such labeling are well-knownin the art, for example, see Stemberger et al., J. Histochem. Cytochem.18: 315, 1970; Bayer et al., Meth. Enzym. 62: 308-, 1979; Engval et al.,Immunol. 109: 129-, 1972; Goding, J. Immunol. Meth. 13: 215-, 1976. Thelabeled antibodies of the present invention can be used for in vitro, invivo, and in situ assays to identify cells or tissues which express aspecific peptide.

The above-described antibodies may also be immobilized on a solidsupport. Examples of such solid supports include plastics such aspolycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir et al., “Handbook of Experimental Immunology” 4th Ed., BlackwellScientific Publications, Oxford, England, Chapter 10, 1986; Jacoby etal., Meth. Enzym. 34, Academic Press, N.Y., 1974). The immobilizedantibodies of the present invention can be used for in vitro, in vivo,and in situ assays as well as in immunochromotography.

Furthermore, one skilled in the art can readily adapt currentlyavailable procedures, as well as the techniques, methods and kitsdisclosed herein with regard to antibodies, to generate peptides capableof binding to a specific peptide sequence in order to generaterationally designed antipeptide peptides (Hurby et al., “Application ofSynthetic Peptides: Antisense Peptides”, In Synthetic Peptides, A User'sGuide, W.H. Freeman, NY, pp. 289-307, 1992; Kaspczak et al.,Biochemistry 28: 9230-9238, 1989).

Anti-peptide peptides can be generated by replacing the basic amino acidresidues found in the peptide sequences of the kinases of the inventionwith acidic residues, while maintaining hydrophobic and uncharged polargroups. For example, lysine, arginine, and/or histidine residues arereplaced with aspartic acid or glutamic acid and glutamic acid residuesare replaced by lysine, arginine or histidine.

The present invention also encompasses a method of detecting aSTE20-related kinase polypeptide in a sample, comprising: (a) contactingthe sample with an above-described antibody, under conditions such thatimmunocomplexes form, and (b) detecting the presence of said antibodybound to the polypeptide. In detail, the methods comprise incubating atest sample with one or more of the antibodies of the present inventionand assaying whether the antibody binds to the test sample. Alteredlevels of a kinase of the invention in a sample as compared to normallevels may indicate disease.

Conditions for incubating an antibody with a test sample vary.Incubation conditions depend on the format employed in the assay, thedetection methods employed, and the type and nature of the antibody usedin the assay. One skilled in the art will recognize that any one of thecommonly available immunological assay formats (such asradioimmunoassays, enzyme-linked immunosorbent assays, diffusion basedOuchterlony, or rocket immunofluorescent assays) can readily be adaptedto employ the antibodies of the present invention. Examples of suchassays can be found in Chard (“An Introduction to Radioimmunoassay andRelated Techniques” Elsevier Science Publishers, Amsterdam, TheNetherlands, 1986), Bullock et al. (“Techniques in Immunocytochemistry,”Academic Press, Orlando, Fla. Vol. 1, 1982; Vol. 2, 1983; Vol. 3, 1985),Tijssen (“Practice and Theory of Enzyme Immunoassays: LaboratoryTechniques in Biochemistry and Molecular Biology,” Elsevier SciencePublishers, Amsterdam, The Netherlands, 1985).

The immunological assay test samples of the present invention includecells, protein or membrane extracts of cells, or biological fluids suchas blood, serum, plasma, or urine. The test samples used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein extracts or membraneextracts of cells are well known in the art and can be readily beadapted in order to obtain a sample which is testable with the systemutilized.

A kit contains all the necessary reagents to carry out the previouslydescribed methods of detection. The kit may comprise: (i) a firstcontainer means containing an above-described antibody, and (ii) secondcontainer means containing a conjugate comprising a binding partner ofthe antibody and a label. In another preferred embodiment, the kitfurther comprises one or more other containers comprising one or more ofthe following: wash reagents and reagents capable of detecting thepresence of bound antibodies.

Examples of detection reagents include, but are not limited to, labeledsecondary antibodies, or in the alternative, if the primary antibody islabeled, the chromophoric, enzymatic, or antibody binding reagents whichare capable of reacting with the labeled antibody. The compartmentalizedkit may be as described above for nucleic acid probe kits. One skilledin the art will readily recognize that the antibodies described in thepresent invention can readily be incorporated into one of theestablished kit formats which are well known in the art.

VI. Isolation of Compounds Which Interact With STE20-Related Kinases

The present invention also relates to a method of detecting a compoundcapable of binding to a STE20-related kinase of the invention comprisingincubating the compound with a kinase of the invention and detecting thepresence of the compound bound to the kinase. The compound may bepresent within a complex mixture, for example, serum, body fluid, orcell extracts.

The present invention also relates to a method of detecting an agonistor antagonist of kinase activity or kinase binding partner activitycomprising incubating cells that produce a kinase of the invention inthe presence of a compound and detecting changes in the level of kinaseactivity or kinase binding partner activity. The compounds thusidentified would produce a change in activity indicative of the presenceof the compound. The compound may be present within a complex mixture,for example, serum, body fluid, or cell extracts. Once the compound isidentified it can be isolated using techniques well known in the art.

The present invention also encompasses a method of agonizing(stimulating) or antagonizing kinase associated activity in a mammalcomprising administering to said mammal an agonist or antagonist to akinase of the invention in an amount sufficient to effect said agonismor antagonism. A method of treating diseases in a mammal with an agonistor antagonist of STE20-related kinase activity comprising administeringthe agonist or antagonist to a mammal in an amount sufficient to agonizeor antagonize STE20-related kinase associated functions is alsoencompassed in the present application.

In an effort to discover novel treatments for diseases, biomedicalresearchers and chemists have designed, synthesized, and testedmolecules that inhibit the function of protein kinases. Some smallorganic molecules form a class of compounds that modulate the functionof protein kinases. Examples of molecules that have been reported toinhibit the function of protein kinases include, but are not limited to,bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO92/20642, published Nov. 26, 1992 by Maguire et al.), vinylene-azaindolederivatives (PCT WO 94/14808, published Jul. 7, 1994 by Ballinari etal.), 1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992),styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridylcompounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EPApplication No. 0 566 266 A1), seleoindoles and selenides (PCT WO94/03427, published Feb. 17, 1994 by Denny et al.), tricyclicpolyhydroxylic compounds (PCT WO 92/21660, published Dec. 10, 1992 byDow), and benzylphosphonic acid compounds (PCT WO 91/15495, publishedOct. 17, 1991 by Dow et al).

Compounds that can traverse cell membranes and are resistant to acidhydrolysis are potentially advantageous as therapeutics as they canbecome highly bioavailable after being administered orally to patients.However, many of these protein kinase inhibitors only weakly inhibit thefunction of protein kinases. In addition, many inhibit a variety ofprotein kinases and will cause multiple side-effects as therapeutics fordiseases.

Some indolinone compounds, however, form classes of acid resistant andmembrane permeable organic molecules. WO 96/22976 (published Aug. 1,1996 by Ballinari et al.) describes hydrosoluble indolinone compoundsthat harbor tetralin, naphthalene, quinoline, and indole substituentsfused to the oxindole ring. These bicyclic substituents are in turnsubstituted with polar moieties including hydroxylated alkyl, phosphate,and ether moieties. U.S. patent application Ser. No. 08/702,232, filedAug. 23, 1996, entitled “Indolinone Combinatorial Libraries and RelatedProducts and Methods for the Treatment of Disease” by Tang et al. and08/485,323, filed Jun. 7, 1995, entitled “Benzylidene-Z-IndolineCompounds for the Treatment of Disease” by Tang et al. and InternationalPatent Publication WO 96/22976, published Aug. 1, 1996 by Ballinari etal., all of which are incorporated herein by reference in theirentirety, including any drawings, describe indolinone chemical librariesof indolinone compounds harboring other bicyclic moieties as well asmonocyclic moieties fused to the oxindole ring. Application Ser. No.08/702,232, filed Aug. 23, 1996, entitled “Indolinone CombinatorialLibraries and Related Products and Methods for the Treatment of Disease”by Tang et al., 08/485,323, filed Jun. 7, 1995, entitled“Benzylidene-Z-Indoline Compounds for the Treatment of Disease” by Tanget al., and WO 96/22976, published Aug. 1, 1996 by Ballinari et al.teach methods of indolinone synthesis, methods of testing the biologicalactivity of indolinone compounds in cells, and inhibition patterns ofindolinone derivatives.

Other examples of substances capable of modulating kinase activityinclude, but are not limited to, tyrphostins, quinazolines,quinoxolines, and quinolines. The quinazolines, tyrphostins, quinolines,and quinoxolines referred to above include well known compounds such asthose described in the literature. For example, representativepublications describing quinazolines include Barker et al., EPOPublication No. 0 520 722 A1; Jones et al., U.S. Pat. No. 4,447,608;Kabbe et al., U.S. Pat. No. 4,757,072; Kaul and Vougioukas, U.S. Pat.No. 5,316,553; Kreighbaum and Corner, U.S. Pat. No. 4,343,940; Pegg andWardleworth, EPO Publication No. 0 562 734 A1; Barker et al., Proc. ofAm. Assoc. for Cancer Research 32: 327 (1991); Bertino, J. R., CancerResearch 3: 293-304 (1979); Bertino, J. R., Cancer Research 9(2 part 1):293-304 (1979); Curtin et al., Br. J. Cancer 53: 361-368 (1986);Fernandes et al., Cancer Research 43: 1117-1123 (1983); Ferris et al. J.Org. Chem. 44(2): 173-178; Fry et al., Science 265: 1093-1095 (1994);Jackman et al., Cancer Research 51: 5579-5586 (1981); Jones et al. J.Med. Chem. 29(6): 1114-1118; Lee and Skibo, Biochemistry 26(23):7355-7362 (1987); Lemus et al., J. Org. Chem. 54: 3511-3518 (1989); Leyand Seng, Synthesis 1975: 415-522 (1975); Maxwell et al., MagneticResonance in Medicine 17: 189-196 (1991); Mini et al., Cancer Research45: 325-330 (1985); Phillips and Castle, J. Heterocyclic Chem. 17(19):1489-1596 (1980); Reece et al., Cancer Research 47(11): 2996-2999(1977); Sculier et al., Cancer Immunol. and Immunother. 23: A65 (1986);Sikora et al., Cancer Letters 23: 289-295 (1984); Sikora et al.,Analytical Biochem. 172: 344-355 (1988); all of which are incorporatedherein by reference in their entirety, including any drawings.

Quinoxaline is described in Kaul and Vougioukas, U.S. Pat. No.5,316,553, incorporated herein by reference in its entirety, includingany drawings.

Quinolines are described in Dolle et al., J. Med. Chem. 37: 2627-2629(1994); MaGuire, J. Med. Chem. 37: 2129-2131 (1994); Burke et al., J.Med. Chem. 36: 425-432 (1993); and Burke et al. BioOrganic Med. Chem.Letters 2: 1771-1774 (1992), all of which are incorporated by referencein their entirety, including any drawings.

Tyrphostins are described in Allen et al., Clin. Exp. Immunol. 91:141-156 (1993); Anafi et al., Blood 82: 12: 3524-3529 (1993); Baker etal., J. Cell Sci. 102: 543-555 (1992); Bilder et al., Amer. Physiol.Soc. pp. 6363-6143: C721-C730 (1991); Brunton et al., Proceedings ofAmer. Assoc. Cancer Rsch. 33: 558 (1992); Bryckaert et al., ExperimentalCell Research 199: 255-261 (1992); Dong et al., J. Leukocyte Biology 53:53-60 (1993); Dong et al., J. Immunol. 151(5): 2717-2724 (1993); Gazitet al., J. Med. Chem. 32: 2344-2352 (1989); Gazit et al., “J. Med. Chem.36: 3556-3564 (1993); Kaur et al., Anti-Cancer Drugs 5: 213-222 (1994);Kaur et al., King et al., Biochem. J. 275: 413-418 (1991); Kuo et al.,Cancer Letters 74: 197-202 (1993); Levitzki, A., The FASEB J. 6:3275-3282 (1992); Lyall et al., J. Biol. Chem. 264: 14503-14509 (1989);Peterson et al., The Prostate 22: 335-345 (1993); Pillemer et al., Int.J. Cancer 50: 80-85 (1992); Posner et al., Molecular Pharmacology 45:673-683 (1993); Rendu et al., Biol. Pharmacology 44(5): 881-888 (1992);Sauro and Thomas, Life Sciences 53: 371-376 (1993); Sauro and Thomas, J.Pharm. and Experimental Therapeutics 267(3): 119-1125 (1993); Wolbringet al., J. Biol. Chem. 269(36): 22470-22472 (1994); and Yoneda et al.,Cancer Research 51: 4430-4435 (1991); all of which are incorporatedherein by reference in their entirety, including any drawings.

Other compounds that could be used as modulators include oxindolinonessuch as those described in U.S. patent application Ser. No. 08/702,232filed Aug. 23, 1996, incorporated herein by reference in its entirety,including any drawings.

VII. Biological Significance, Applications and Clinical Relevance ofNovel STE20-Related Kinases

Human STLK2, STLK3, STLK4, STLK5, STLK6, and STLK7

STLK2, STLK4, STLK5, STLK6 and STLK7 belong to an expanding family ofintracellular STKs that have varying degrees of sequence homology toSOK-1, a kinase implicated in oxidative stress agents (Pombo, C M et al,EMBO J. (17) 4537-4546, 1996). Our data shows that STLK2 is expressedhighly in hematopoietic cells. Therefore, STLK2 may participate in theoxidative response pathway during inflammation. In addition, STLK2 couldalso be a possible component in the signaling pathways leading to T cellactivation. High levels of STLK2 in several tumor cell lines could alsoimply that STLK2 might be involved in tumorigenesis.

STLK2 is most closely related to two human STE20-subfamily kinases: MST3and SOK-1. MST3 is a 52,000 daltons cytoplasmic kinase that isubiquitously expressed with its highest levels of expression found inheart, skeletal muscle and pancreas. The serine/threonine kinaseactivity of MST3 is activated by phosphorylation. Unlike SOK-1, MST3prefers Mn⁺⁺ over Mg⁺⁺ and can use both GTP and ATP as phosphate donors.MST3 may undergo dimerization. No agonists have yet been identified thatactivate MST3. The downstream signaling mechanism of this kinase isunknown (Schinkmann, K and Blenis, J. (1997) J. Biol. Chem. 272,28695-28703).

SOK-1 is a 50,000 daltons cytoplasmic kinase expressed predominantly intestis, large intestine, brain and stomach and to a lesser extent inheart and lung. SOK-1 is also expressed in the germinal center B-cellline (RAMOS) and in a mature B cell line (HS Sultan). Theserine/threonine kinase activity of SOK-1 is activated byphosphorylation. The C-terminus of SOK-1 has been shown to be inhibitoryto the catalytic activity of this kinase. The only agonists known toactivate SOK-1 are oxidant agents, like H₂O₂ and menadione, a quinonethat is a potent intracellular generator of reactive oxygen species(Pombo, C. M. et al. EMBO J. 15, 4537-4546). SOK-1 is also activated bychemical anoxia through the generation of reactive oxygen species andrelease of calcium into the cytoplasm from intracellular stores. SOK-1,therefore, may play an important role in ischemia, the cause ofmyocardial infarction, stroke and acute renal failure (Pombo, C. M. etal. J. Biol. Chem. 272, 29372-29379 (1997)). The activity of SOK-1 inthe response to oxidant stress is inversely correlated with the activityof the stress-activated protein kinases (SAPKs): elevated SOK-1 activitycorrelates with absent SAPK activity and vice-versa. SOK-1 does notactivate any of the four MAP kinase pathways, SAPKs, p38, ERK-1 orMEK-5/ERK-5 (Pombo, C. M. et al. EMBO J. 15, 4537-4546). The downstreamsignaling mechanism of this kinase remains unknown.

STLK2 is expressed in a wide variety of immune cell types and tissuesincluding thymus, dendrocytes, mast cells, monocytes, B cells (primary,Jurkat, RPMI, SR), T cells (CD8/CD4+, TH1, TH2, CEM, MOLT4) andmegakaryocytes (K562), whereas STLK3 is restricted to thymus and STLK4is predominately expressed in thymus, T cells (CD4/CD8+, TH1, CEM) and Bcells (Jurkat, RPMI). Consequently, these STKs might participate in theoxidative response pathway during inflammation, reperfusion injury(stroke, surgery, shock), TNFα-mediated signaling, insulindesensitization, atherogenesis, vascular injury, T or B cellcostimulation, or alternatively, participate in other MAPK-relatedsignal transduction processes.

STLK5 is more distantly related to this STE20-subfamily including SOK-1and STLK2, STLK3 and STLK4. STLK5, may therefore mediate a signalingpathway that is distinct from the oxidative stress response pathway.

The high degree of sequence homology in the C-termini of SOK-1, STLK2,STLK3, STLK4, STLK5, and STLK6 raises the possibility that these novelSTKs, like SOK-1, may be subject to autoinhibition through a conservedC-terminal motif.

Human ZC1, ZC2, ZC3 and ZC4

ZC1 is a good candidate for any disease in which tyrosine kinase,cytokine, or heterotrimeric G-protein coupled receptors have beenimplicated. The mouse homologue binds to NCK, and is recruited toactivated PDGF (Su et al., EMBO 16: 1279-1290, 1997). The Drosophilahomolog has been shown to bind to TRAF2, implicating it in TNF-αsignaling (Liu et al., (1999) Curr. Biol. 9: 101-104, 1999)). While ZC1does not contain the exact NCK- and TRAF2-binding domains, it is likelyto bind to related proteins.

Of the ZC subfamily of STE20-related protein kinases, ZC1 has very broadover-expression in many tumor types, suggesting that it may be involvedin cellular growth, transformation, or tumor progression. A truncatedform of ZC1 containing only the C-terminal putative MEKK1-binding domainwas found to reduce the number of foci generated by H-Ras-V12 in RatIntestinal Epithelial cells (RIE-1). These data indicate that ZC1 mayplay a role in the ability for these cells to overcome contactinhibition and anchorage-dependent growth.

The ZC1 homolog, Misshapen (msn) in Drosophila melanogaster was clonedas a result of complementing a mutation in a developmental pathwayrequired for dorsal closure, a process involving changes in cell shapeand position in the embryo (Treisman et al. Gene 186 119-125, 1997). AD. melanogaster homolog of the JNK1/JNK2 kinases from mammals was shownto function downstream of msn in the dorsal-closure signaling pathway(Su et al. Genes Dev. 12: 2371-2380, 1998).

While ZC1 could be involved in multiple aspects of tumorigenesis, byanalogy with Drosophila, the role of misshapen in dorsal closuresuggests a critical role in the regulation of the cytoskeleton for theprocesses of cell attachment, cell movement and perhaps migration.

The association of the ZC1 family members msn and NIK with TRAF2 mayindicate a role for this kinase in cell survival and/or in apoptosis.The ZC1 family contains a highly conserved domain that in the mousehomolog, NIK, has been shown to bind to MEKK1(Mitogen-activated/Extracellular-regulated Kinase Kinase 1) (Su et al.,(1997) EMBO 16(6): 1279-90). MEKK1 is involved in cell survival and/orapoptosis in several systems (Schlesinger et al., Front. Biosci. 3:D1181-6, 1998). Depending on the context, MEKK1 appears to be upstreamof either the ERK1/MAPK or the JNK/SAPK pathway [Schlesinger et al.,(1998 Front. Biosci. 3: D1181-6). Three homologues of ZC1: murine NIK(NCK-interacting kinase)(Su et al. EMBO 16: 1279-90, 1997), Drosophilamsn (Liu et al. Curr. Biol. 9: 101-104, 1999) and human HGK(HPK/GCK-like kinase)(Yao et al., J. Biol. Chem. 274: 2118-25, 1999)have all been shown to activate the JNK pathway when over-expressed in293T cells.

ZC1 shares a high degree of homology with these other family members inboth the kinase domain and the “MEKK”-binding domains, yet it differs inthe intervening region, which contains several putative binding domainsfor upstream signaling adapter molecules (e.g. NCK, TRAF2). Unlike theother family members, ZC1 does not appear to activate the JNK pathway in293T cells as seen by its ability to induce expression of either a JUNor ATF2-driven luciferase gene. Upon co-transfection into these cellswith HA-tagged JNK, modest activation of JNK was detected. ZC1 alsomodestly activated co-transfected ERK1. Both the ERK and the JNKactivation were very slight compared with the positive controls in theassay (activated forms of MEK1 and MEKK1, respectively). In both cases,activation required the full-length kinase. While the kinase domainalone is up to 5× more active in autophosphorylation and inphosphorylation of MBP, it does not lead to activation of thesepotential downstream kinases. Based on the strong sequence homology ofZC1 with the other family members, it is very likely that ZC1 will beimportant for either JNK or ERK activation once the proper context isfound.

ZC1 profoundly inhibits ERK1 kinase expression in co-transfectionassays. This effect is dependent on ZC1 kinase activity, occurring withthe wild-type and the kinase domain alone, but not with the kinase-deadmutant even though all three forms of ZC1 are expressed at similarlevels. This may suggest a role for this kinase in transcriptional orpost-transcriptional regulation.

ZC1 may be an important component in the signaling pathways mediated bythe co-stimulatory receptor CD28 in T cells and/or by thepro-inflammatory cytokine TNFα, since co-transfection of the wild-typeZC1 activated the RE/AP-luciferase and NFκB-luciferase reporter genes.While our data showed that ZC1 strongly activates NFκB in T-cells, noactivation of NFκB driven luciferase was detectable in NIH 3T3 cells. Arecent paper (J. Biol. Chem. 274: 2118-25; 1999.) has shown that a humanZC1 splicing isoform, HGK, is involved in the TNFα-signaling pathways.

Given the importance of T cell activation in autoimmunity andtransplantation, as well as the key role that TNFα plays in inflammatorydiseases, it is possible that ZC1 could be a therapeutic target forimmunological diseases which include but are not limited to: rheumatoidarthritus, chronic inflammatory bowel diseases (ie Crohn's disease),chronic inflammatory pelvic disease, multiple sclerosis, asthma,osteoarthritis, psoriasis, atherosclerosis, rhinitis, and autoimmunityas well as organ transplantation and cardiovascular diseases.

ZC1 appears to be the human orthologue of murine NIK and possibly anorthologue of a C. elegans STE20-subfamily kinase encoded by the ZC504.4cosmid.

Murine NIK is a 140,000 daltons kinase that is most highly expressed inbrain and heart. NIK interacts with the SH3 domains of the adaptormolecule Nck through its proline-rich regions found in the C-terminalextra-catalytic region. The specific regions that mediate thisinteraction are two PxxP (SEQ ID NO: 148) motifs that are nearlyuniformly conserved between NIK, ZC1,2,3 and the C. elegans STE20ZC504.4 kinase. In addition, NIK binds MEKK1 through its 719 amino acidC-terminal (Su, Y-C. et al. (1997) EMBO J. 16, 1279-1290). MEKK1 is amembrane-associated kinase responsible for activating MKK4 (also knownas SEK1), which in turn activates SAPK (Yan, M et al. (1994) Nature,372, 798-800). NIK may function as a kinase that links growth factoractivated pathways and the stress-response pathway mediated by SAPKs.According to this hypothesis, activation of growth factor receptorsleads to receptor tyrosine phosphorylation, Nck binding to thephosphorylated tyrosines via its SH2 domain, NIK redistribution to amembrane compartment via binding to the SH3 domain of Nck, andjuxtaposition to the membrane-associated MEKK1. The NIK-MEKK1interaction would, in this fashion, turn on the SAPK pathway in responseto growth factor stimulation (Su, Y-C. et al. (1997) EMBO J. 16,1279-1290).

Given the high homology between ZC1, ZC2, ZC3, and ZC4 STKs and NIK, itis conceivable that these kinases may each function to connect growthfactor- and stress-activated signaling pathways. The heterogeneity thatthe ZC kinases exhibit within their putative SH3-binding domain couldprovide signaling specificity in terms of the nature of the adaptormolecule that they bind. The high level of sequence conservation in theC-termini of the ZC1, ZC2 and ZC3 strongly suggests that these humankinases, like murine NIK, also may bind to MEKK1 and activate SAPKs. TheZC kinases also display strong homology at their C-termini to proteindomains that bind small GTPase proteins such as Rab, Rho and Rac. Forexample, the C-termini of ZC1 is 36.2% identical to citron, a murineRho-binding protein, and 23.1% identical to the rab-binding region of GCkinase. This suggests that, in addition to adaptor molecules, smallGTPase proteins may also mediate membrane association and activation ofthe ZC kinases. The presence of a potential coiled-coil region locatedimmediately C-terminal to the catalytic region strongly suggests thatthe ZC kinases may also be subject to regulation via homo orheterodimerization events.

The C. elegans STE20 ZC504.4 kinase is the product of the mig-15 gene.The product of this gene has been implicated in several developmentalprocesses such as epidermal development, Q neuroblast migrations andmuscle arm targeting in the developing worm (Zhu, X. and Hedgecock E.(1997) Worm Breeder's Gazette 14, 76). The high level of sequenceconservation between the ZC kinases and the ZC504.4 C. elegans kinasewill make C. elegans a valuable model organism to study, throughepistatic analysis, the signaling properties of the human ZC kinases.

Human KHS2

KHS1 (kinase homologous to SPS1/STE20) is a 100,000 dalton cytoplasmicSTK that is expressed ubiquitously. KHS 1 has been implicated in themechanism of SAPK activation in response to inflammatory cytokines suchas TNF□ as well as to ultraviolight light, which also uses the TNFsignaling pathway. TNF□ binding to its receptors (TNFR1 and TNFR2)results in the sequential association with the receptor C-tail ofmultiple signaling molecules including TNFR1-associated death domainprotein (TRADD), Fas-associated death domain protein (FADD or MORT1),TNFR-associated factor 2 (TRAF2), and the STK RIP (receptor interactingprotein). The TRADD-TRAF2 interaction is mediated by a conserved regionpresent at the C-terminus of TRAF2, the TRAF domain. Activation of theNF□B and SAPK pathways is mediated by the ring finger motif present atthe N-terminus of TRAF2 (Curr. Opinion in Cell. Biol. (1997) 9:247-251). KHS1 is activated by TNFα stimulation in a TRAF2-dependantmanner and inhibition of KHS1 blocks TNFα-induced SAPK activation butnot NFOB activation. The mechanism by which TRAF2 activates KHS1 is notknown. Cotransfection of TRAF2- and KHS1-expressing constructs in 293Tcells failed to reveal a direct association between these two molecules.KHS1 activates the SAPK pathway by a direct association with theconstitutively active kinase MEKK1. MEKK1 subsequently activates SEK1,which in turn activates SAPK. Neither the MAPK nor the p38 kinasepathways are activated by KHS1 (Shi, C-S and Kehrl. J. H. (1997) J.Biol. Chem. 272, 32102-32107). In addition to its catalytic domain,downstream signaling of KHS1 requires its conserved C-terminus (Diener,K. et al (1997) Proc. Natl. Acad. Sci. 94, 9687-9692).

GCK (germinal center kinase) is a constitutively active 97,000 daltonSTK that is broadly expressed. GCK may participate in B-celldifferentiation since its expression is localized to the germinal centerwithin lymphoid follicles. GCK activates the SAPK pathway in response toTNFα via activation of SEK1. The upstream activators of GCK in responseto cytokines as well as the immediate downstream target of this kinaseare unknown. The C-terminus of GCK is sufficient to activate SEK1(Pombo, C. M. et al (1995) Nature, 377, 750-754).

The murine orthologue of GCK, rab8ip (rab8-interacting protein), is a97,000 dalton protein that fractionates with both the solublecytoplasmic fraction as well as with a salt-sensitive fractionassociated with the basolateral membrane of the trans-Golgi region inpolarized MDCK epithelial cells. The C-terminus of rab8ip binds to rab8,a small GTP-binding protein required for vesicular transport from theGolgi apparatus (Ren, M. et al. (1996) Proc. Natl. Acad. Sci. 93,5151-5155). In addition to inducing the transcriptional activation ofcytokines like IL2 via SAPK, GCK may also promote the rab-dependentrelease of secretory proteins in response to TNFα (Buccione, R. et al(1995) Mol. Bio. Cell 6, 291).

HPK1 (hematopoietic protein kinase) is a constitutively active 90,000dalton STK restricted to hematopoietic cells. HPK1 activates the SAPKpathway by directly binding to and activating MEKK1 (Hu, M. et al (1996)Genes and Dev. 10: 2251-2264) as well as the ubiquitously expressedmixed-lineage kinase MLK-3 (Kiefer, F. et al (1996) EMBO J. 15:7013-7025). This function of HPK1 requires, in contrast to GCK, both itskinase domain as well as its C-terminus. The upstream activators of HPK1remain unknown. HPK1 also plays a key role as a mediator of transforminggrowth factor-β-(TGFβ) signaling. HPK1 activates the TGFb-activatedkinase (TAK), which in turn stimulates the SAPK pathway byphosphorylating SEK1 (Wang W. et al (1997) J. Biol. Chem. 272:22771-22775).

KHS2 is expressed in thymus, dendrocytes and monocytes. KHS2 could havea complementary function to that of KHS 1 as a mediator of SAPKactivation in the cellular response to inflammatory cytokines. KHS2could have the potential to interact directly with TRAF2 since a STKwith the predicted molecular weight of KHS2 (approximately 101,000daltons) is found in the TNFR-TRAF2 complex upon TNFβ stimulation(VanArsdale, T. and Ware, C. F. (1994) J. Immunol. 153, 3043-3050). Thepresence of a putative binding domain for Rab or a Rab-like molecule atthe C-terminus of KHS2 indicates that KHS2, in addition to having apotential role in the TRAF2-dependant TNFα cytokine response, could alsomediate signaling events that utilize small GTPase proteins.Alternatively, the binding of a small GTPase protein to the C-terminusof KHS2 may be required for its potential TRAF2-dependant signaling to adownstream kinase such as MEKK1.

Human GEK2, SULU1 and SULU3

A recent report (Y-W Qian et al., Science 282: 1701-1704,1998) describedxPlkk1 as the activator of Plx1 (the Xenopus Polo kinase). In Xenopusoocytes, the STK Plkk1 can phosphorylate and activate Plx1 STK (themammalian Polo kinase or PLK). A dominant-negative (kinase-dead) form ofxPlkk1 prevents Plx1 activation and delays germinal vesicle breakdown.Yet another unidentified kinase is probably responsible for xPlkk1activation during mitosis.

The homology through the entire length of the xPlkk1 protein with GEK2suggests that GEK2 might represent the human homologue for xPlkk1. Basedon this, GEK2 might be upstream of PLK in mammalian cells. In addition,based on the phage display screen results using the SULU1 coiled-coil2domain as bait, SULU1 might also interact in vivo with GEK2 andtherefore regulate GEK2 (and/or SLK through the coiled-coil domain)activation leading to PLK activation and mitosis.

If such a cascade of events is required for mitosis in mammalian cells,interruption of this signaling cascade at any point might block mitosisand could be beneficial for cancer treatment.

A recently cloned STE20-subfamily kinase, rat TAO1, is most likely therodent orthologue of human SULU3 (Hutchinson, M. et al. J. Biol. Chem273: 28625-28632, 1998). TAO1 activates MEK3, 4 and 6 in vitro, while intransfected cells it associates and activates only MEK3, resulting inphosphorylation and activation of p38. These results implicate TAO1(SULU3) in the regulation of the p38 containing stress-responsive MAPkinase pathway.

Human SULU1 is weakly expressed in hematopoietic sources whereas SULU3is found in B-cells and TH1-restricted T cells. These mammalian SULUSTKs display strong homology to the C. elegans SULU kinase. The rolethat this kinase plays in nematode development is unknown. The strongsequence homology between the catalytic domain of mammalian SULU kinasesand other STE20-subfamily kinases such as SOK-1 (human STE20) and KHS2suggests that the mammalian kinases may participate in thestress-response pathway. The potential coiled-coil domains found at theC-terminus of the SULU kinases may play a role in the regulation of thiskinase.

Murine LOK (lymphocyte-oriented kinase) is a constitutively activatedSTK of approximately 130,000 daltons that is predominantly expressed inspleen, thymus and bone marrow (Kuramochi, S. et al (1997) J. Biol.Chem. 272: 22679-22684) as well as in meiotic testicular and primordialgerm cells. The LOK1 gene is located in chromosome 11 of the mouse nearthe wr locus, a region that is associated with reproductive andneurological defects (Yanagisawa, M. et al (1996) Mol. Reprod. and Dev.45: 411-420). LOK does not activate any of the known MAPK pathways (ERK,JNK and p38) nor the NFkB pathway. The upstream signaling elements ofLOK as well as the extracellular stimuli that utilize this kinase toelicit a biological response are also unknown (Kuramochi, S. et al(1997) J. Biol. Chem. 272: 22679-22684).

Human GEK2 is highly related to murine LOK, but based on sequencedivergence in the non-catalytic domain, it appears to be a distinctmember of this STE20-subfamily. GEK2 may signal through a pathway thatremains to be defined. The presence of potential coiled-coil regions atthe C-terminus of GEK2 could play a key role in regulating the functionsof this kinase.

Human PAK4 and PAK5

The p21 activated protein kinases (PAK) are a closely related subgroupof the STE20 family of serine/threonine kinases. Extensive genetic andbiochemical analysis of the budding yeast STE20 has shown the criticalrole this serine/threonine kinase plays at the juncture of severalimportant intracellular pathways required to appropriately respond toextracellular signals. STE20 links the transcriptional response bymediating the activation of the appropriate downstream MAPK pathway aswell as coupling changes in cellular morphology via its control of theactin cytoskeleton.

A hallmark of the PAK subgroup is their small G protein-binding domain(PBD) that confers G protein-dependent activation upon this group ofkinases. Via the PBD, PAKs bind to activated small G proteins resultingin the derepression of the PAK's intrinsic kinase activity.

Until recently, there were three known PAK kinases: PAK1, a 68 kDprotein whose expression is restricted expression to brain, muscle, andspleen; PAK2 (PAK1, PAK65), a 62 kD protein whose expression isubiquitous; and PAK3, a 65 kD protein whose expression is restricted tothe brain. Similar to STE20, the mammalian PAKs (1,2, and 3) have beenshown to respond to extracellular signals (growth factors, mitogens,cytokines and a variety of cellular stresses) (Bagrodia, et al. (1995).J. Biol. Chem. 270: 22731-22737; Zhang, S., et al. (1995). J. Biol.Chem. 270: 23934-23936, Frost, J. et al. (1998) J. Biol. Chem. 273:28191-28198; Galisteo, M. et al. (1996) J. Biol. Chem. 271:20997-21000), and are linked to TCR activation (Yablonski, D., et al.(1998) EMBO J. 17: 5647-5657), and heterotrimeric G protein-coupledreceptors (Knaus, U. et al. (1995) Science 269: 221-223).

The PAKs were originally identified as effectors for members of the Rhofamily of small G proteins (such as Rac and Cdc42), hence their name,p21-activated kinases (PAK) (Manser et al Nature 367: 40-46). Therecruitment of the PAKs to the appropriate intracellular location iscritical to their function. Attempts to elucidate the role played byPAKs in intracellular signaling and morphological changes is complicateddue to the complex interactions by which they can be recruited by suchfactors as activated small G proteins (rac, cdc42), adaptors (nck) andexchange proteins (PIX, Cool).

The adaptor molecule, Nck, is constitutively bound via its SH3 domain tothe proline-rich motif in the N-terminal portion of PAK1. Binding of theNck-PAK complex to activated growth factor receptors in response togrowth factor stimulation provides a mechanism to link growthfactor-stimulated and stress-response pathways (Galisteo, M. et al.(1996) J. Biol. Chem. 271: 20997-21000).

The PBD found at the N-terminus of PAK1 is responsible for itshigh-affinity interaction with the GTP-bound forms of Cdc42 and Rac(Burbelo, P. et al. (1995) J. Biol. Chem. 270: 29071-29074). The exactmechanism through which the small GTPases activate PAKs may involve, inpart, association of the kinase with activated growth factor receptorsthrough guanine nucleotide exchange factors (GEFs). GEFs activate smallGTPases by catalyzing the formation of their GTP-bound state, therebypromoting their association with, and activation of, PAKs. The knownmammalian PAK kinases, as well as Drosophila and C. elegans PAKs, allconserve an N-terminal extracatalytic motif responsible for ahigh-affinity interaction with the GEF, PIX. The PAK-Cdc42 interactionand subsequent PAKs occurs as a PIX/PAK complex (Manser, E. et al.(1998) Molecular Cell, 1, 183-192).

PAK signaling stimulated by heterotrimeric G proteins is mediatedthrough the interaction between a short conserved amino acid regionlocated at the C-terminus of PAK1 with the G-protein β-subunit (Leeuw,T. et al. (1998) Nature, 391: 191-195).

A variety of studies have indicated that the human PAKs are involved inmediating the activation of stress-activated protein kinase pathways(JNK and to lesser extent p38). PAKs are also potential mediators in thecrosstalk between the pathways regulated by the Rho family of small Gproteins and the signaling pathways directly downstream of Ras leadingto the activation of the ERK pathway (Bagrodia, et al. (1995). J. Biol.Chem. 270: 22731-22737; Zhang, S., et al. (1995). J. Biol. Chem. 270:23934-23936; Brown, J., et al. (1996) Curr Biol. 6: 598-60596; Frost,J., et al. (1996). Mol. Cell. Biol. 16: 3707-3713).

PAK1 has been implicated in phosphorylating a regulatory site in MEK1that is necessary for MEK1's ability to interact with Raf1 (Frost, etal. (1997) EMBO J. 16: 6426-6438). PAK3 has been shown to phosphorylateRaf1 on a site that is important for Raf1 activity (King, A., et al.(1998). Nature 396: 180-183).

PAKs play an important role in controlling morphological changes in cellshape mediated by the actin cytoskeleton. Such morphological changes arerequired for cellular functions ranging from cell division andproliferation to cell motility and vesicle transport. PAK activity hasbeen implicated in the localized assembly (leading edge) and disassembly(retracting edge) of focal adhesions necessary for cell motility (FrostJ. et al (1998) J. Biol. Chem. 273: 28191-28198).

PAK2 may have a role in the morphological changes induced duringapoptosis (Membrane and morphological changes in apoptotic cellsregulated by caspase-mediated activation of PAK2. (Rudel, T. (1997)Science. 276: 1571-4)), and PAK1 may be important in preventingapoptosis (Faure S, et al. (1997) EMBO J. (1997) 16: 5550-61). Inaddition to overcoming mitogen- and anchorage-independent growth, tumorcells need to escape the programmed cell death that accompaniesderegulated cell growth. Thus, inhibition of PAKs may be effective intriggering apoptosis in tumors.

A direct requirement for PAKs in the transformation of mammalian cellshas been shown for PAK1 and PAK2. Kinase-dead alleles of PAK1 block rastransformation of RAT1 and Schwann cells (Tang, Y., et al. (1997) Mol.Cell. Biol. 17, 4454-4464). Dominant-negative alleles of PAK2 have beenshown to interfere with ras-mediated transformation of mammalian cells(Osada, S., (1997) FEBS Lett 404: 227-233).

Mutations in PAK3 have been implicated in nonsyndromic X-linked mentalretardation suggesting a role for PAK3 in cognitive function (Allen, K.et al. (1998) Nat. Genet. 20: 25-30). PAK1 has been implicated inneurite outgrowth in PC12 cells (Daniels, R. et al. (1998) EMBO J. 17:754-764; Nikolic, M. et al. (1998) Nature 395: 194-198).

Finally, PAK-like STKs may also play a role in AIDS pathogenesis sincethe myristoylated 27 kD membrane-associated HIV Nef gene productdirectly interacts with and activates these kinases via cdc42 and Rac.The Nef-mediated activation of PAK-like STKs correlates with theinduction of high viral titers and the development of AIDS in infectedhosts (Cullen, B. R. (1996) Curr. Biol. 6: 1557-1559).

Our results show that PAK4 is expressed in thymus, dendrocytes, mastcells, monocytes, as well as in T cells (TH2-restricted cells and MOLT4)and the B cell line RPMI. PAK5 is found in mast cells and in the T cellline MOLT4. These data suggest potential roles for PAK4 and PAK5 in theimmune system.

PAK4 and PAK5 share with the known PAKs a potential cdc42-binding motifat their N-termini. Both PAK4 and PAK5 display sequence homology intheir C-termini to a motif responsible for an interaction between PAK1and the β-subunit of heterotrimic G-proteins (amino acid residues665-676 in PAK 4, and amino acid residues 386-398 in PAK5).Consequently, PAK4, and possibly PAK5, could mediate signaling eventsoriginating from growth factors as well as from ligands that stimulateG-protein-linked receptors.

PAK4 conserves a leucine (leu 44), that when mutated to a phenylalaninerenders the kinase activity of human PAK1 constitutively active,bypassing its cdc42-binding requirement for activation (Brown J. et al(1996) Current Biol. 6: 598-605). PAK5 contains an isoleucine at theequivalent position. Therefore, the mechanism by which cdc42 potentiallyactivates human PAK1, PAK4, and possibly PAK5, may be very similar.

PAK4 and PAK5 however, lack the PIX-binding motif, and consequentlycdc42-activating GEFs other than PIX (for example Dbl and Cool) must beresponsible for the activation of these kinases. Alternatively, PAK4 andPAK5 may be activated by another GTPase, such as Rac1 which uses theTiam1 GEF for its activation to the GTP-bound state.

PAK4 and PAK5 also lack the PxxP (SEQ ID NO: 148) motif responsible forthe Nck-PAK1 association. Between the PBD or cdc42-binding N-terminalmotifs and the putative GEF-binding regions, PAK4 and PAK5 have longinsertions (185 and 123 amino acids for PAK4 and PAK5, respectively)relative to PAK1. This region probably confers different bindingcharacteristics to adaptor molecules and/or GEFs from those exhibited byknown mammalian PAKs.

PAKs have been shown to be upstream in pathways leading to activation ofboth the JNK (Bagrodia, S., et al. (1995) J. Biol. Chem. 270:22731-22737) and ERK kinase pathways (Brown, J., et al. (1996). CurrBiol. 6: 598-605). PAK1 was shown to synergize with ras in activation ofthe ERK pathway through phosphorylation of MEK1 (Frost, J. et al.(1997). EMBO J. 16: 6426-6438). Our data shows that MEK1 serves as an invitro substrate for PAK4, suggesting a potential role for PAK4 in theactivation of the ERK pathway and mitogenesis.

PAK5 may also have a mitogenic role, and be linked to cancer, based onits expression profile (elevated RNA and protein levels in a widevariety of tumor cell lines), its interaction with cdc42 via its PBD,and the ability of a kinase-dead allele (Lys350, 351 Ala) to block rastransformation of NIH3T3 cells. Thus, a screen for small moleculeinhibitors of PAK5 kinase activity may yield compounds with therapeuticpotential for intervention in cancer derived from a wide variety oftissue types.

PAK4 and PAK5 may also play a role in HIV pathogenesis as potentialmediators of Nef signaling, since none of the known PAKs correspond tothe PAK-like kinase shown to interact with, and be activated by, the HIVnef protein (Lu, X. et al. (1996) Current Biology 6: 1677-1684).

The 3′ untranslated region of PAK4 contains a CA repeat that is prone toundergo expansion. CA dinucleotide repeat instability has beenassociated with disease (Toren, M. Z. et al (1998) Am. J. Hematol. 57:148-152), and expansion of such repeat in the 3′ untranslated region ofPAK4 could implicate this kinase in as yet unknown pathologies.

Clinical Applications

Human STLK2, STLK3, STLK4, STLK5, STLK6, and STLK7

STLK3, STLK5, STLK6 and STLK7, as well as other homologues of the STLKsubfamily of STE20 protein kinases such as STLK4, may play an importantrole as mediators of the immune response. Thus, they are targets for thedevelopment of specific small molecule inhibitors to treat immunologicaldiseases, including, but not limited to, rheumatoid arthritis, chronicinflammatory bowel diseases (e.g. Crohn's disease), chronic inflammatorypelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis,atherosclerosis, rhinitis and autoimmunity, as well as in organtransplantation. Other diseases include cardiovascular diseases.

The human STLKs may also play an important role in cell growthregulation. Thus, they are targets for developing small molecule kinaseinhibitors for the treatment of cancer and metastases. STLK5 maps to achromosomal region frequently amplified in a variety of tumors includingthose from non-small cell lung cancer, breast cancer and peripheralnerve tumors. This suggests that STLK5 could play a role in thedevelopment, maintenance, or progression of human tumors.

The potential role of human STLKs 2, 3, and 4 in mediating oxidativestress strongly suggests that drugs targeting these kinases could proveuseful in the treatment of myocardial infarction, arrhythmia and othercardiomyopathies, stroke, renal failure, oxidative stress-relatedneurodegenerative disorders such amyotrophic lateral sclerosis,Parkinson's disease and Leigh syndrome, a necrotizing mitochondrialencephalopathy, as well.

Human ZC1, ZC2, ZC3, and ZC4

ZC1 may be a component of the CD28-signaling pathway and thereforeimportant in T cell activation. As such, ZC1 as well as other ZCsubfamily kinases, are targets for the development of specific smallmolecule inhibitors to treat immunological diseases, including, but notlimited to, rheumatoid arthritis, chronic inflammatory bowel diseases(e.g. Crohn's disease), chronic inflammatory pelvic disease, multiplesclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitisand autoimmunity, as well as organ transplantation. Other diseasesinclude cardiovascular diseases.

ZC1 and ZC2 are also implicated in cell growth regulation. Thus, ZCsubfamily kinases are targets for developing small molecule inhibitorsfor the treatment of cancer and metastases. ZC2 maps to a chromosomalregion frequently amplified in a variety of tumors including those fromnon-small cell lung cancer, small cell lung cancer, and cervical cancer.This suggests that ZC2 could play a role in the development,maintenance, or progression of human tumors.

The role of human ZC1, ZC2, ZC3, and ZC4 in the inflammatory andstress-response pathways, strongly suggests that drugs targeting thesekinases could have strong immunosuppressive actions. These drugs canprove valuable for the treatment of rheumatoid arthritis,artherosclerosis, autoimmune disorders and organ transplantation amongothers. At least one very important class of immunosuppresants,corticosteroids, functions by blocking SAPK activation at an as yetundefined site on this pathway (Swantek, J. L. et al (1997) Mol. Cell.Biol. (1997) 6274-6282). Other immunosuppresive drugs like the pyridinylimidazoles specifically target the p38 kinases (Kumar, S. et al (1997)Biochem. Biophys. Res. Commun. 235: 533-528). Drug targeting of the MAPKand p38 pathways could lead to the development of novelimmunosuppresants.

Human SULU and GEK

The potential role of these novel STE20-related protein kinases in thecontrol of mitosis strongly suggests that agents that specificallyinhibit these kinases could be useful for cancer and metastasestreatment.

The close homology of human STLK5, GEK2, SULU1 and SULU3 toSTE20-subfamily kinases involved in the stress and oxidative responsepathway strongly suggests that drugs targeting these kinases may also beuseful as immunosuppressants as well as to treat ischemic disorders.

Human KHS2

The role of human KHS2 in the inflammatory and stress-response pathways,strongly suggests that drugs targeting this and related kinases couldhave strong immunosuppressive actions. These drugs can prove valuablefor the treatment of rheumatoid arthritis, artherosclerosis, autoimmunedisorders and organ transplantation among others. At least one veryimportant class of immunosuppresants, corticosteroids, functions byblocking SAPK activation at an as yet undefined site on this pathway(Swantek, J. L. et al (1997) Mol. Cell. Biol. (1997) 6274-6282). Otherimmunosuppresive drugs like the pyridinyl imidazoles specifically targetthe p38 kinases (Kumar, S. et al (1997) Biochem. Biophys. Res. Commun.235: 533-528). Drug targeting of the MAPK and p38 pathways could lead tothe development of novel immunosuppressants.

Human PAK Family

PAK5 has a role in cancer based on its expression profile (elevated RNAand protein levels in wide variety of tumor lines), its interaction withCdc42 via its PBD, and the ability of the kinase-dead allele of PAK5(Lys350, 351Ala) to block ras transformation of NIH3T3 cells. Thus, ascreen for small molecule inhibitors of PAK5 kinase activity may yieldcompounds with therapeutic potential for intervention in cancers andmetastases derived from a wide range of tissue types.

PAK5 maps to a chromosomal region frequently amplified in a variety oftumors including those from non-small cell lung cancer, and small celllung cancer. These findings suggest that PAK5 could play a role in thedevelopment, maintenance, or progression of human tumors and/ormetastases.

The role of human PAK4, and PAK5 in the inflammatory and stress-responsepathways also strongly suggests that drugs targeting these kinases couldhave strong immunosuppressive actions. These drugs can prove valuablefor the treatment of rheumatoid arthritis, artherosclerosis, autoimmunedisorders and organ transplantation among others. At least one veryimportant class of immunosuppresants, corticosteroids, functions byblocking SAPK activation at an as yet undefined site on this pathway(Swantek, J. L. et al (1997) Mol. Cell. Biol. (1997) 6274-6282). Otherimmunosuppresive drugs like the pyridinyl imidazoles specifically targetthe p38 kinases (Kumar, S. et al (1997) Biochem. Biophys. Res. Commun.235: 533-528). Drug targeting of the MAPK and p38 pathways could lead tothe development of novel immunosuppresants. In addition, drugs targetingPAK4 or PAK5 could prove useful as immunosuppresants as well as in AIDStreatment.

VIII. Transgenic Animals.

A variety of methods are available for the production of transgenicanimals associated with this invention. DNA can be injected into thepronucleus of a fertilized egg before fusion of the male and femalepronuclei, or injected into the nucleus of an embryonic cell (e.g., thenucleus of a two-cell embryo) following the initiation of cell division(Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442, 1985).Embryos can be infected with viruses, especially retroviruses, modifiedto carry inorganic-ion receptor nucleotide sequences of the invention.

Pluripotent stem cells derived from the inner cell mass of the embryoand stabilized in culture can be manipulated in culture to incorporatenucleotide sequences of the invention. A transgenic animal can beproduced from such cells through implantation into a blastocyst that isimplanted into a foster mother and allowed to come to term. Animalssuitable for transgenic experiments can be obtained from standardcommercial sources such as Charles River (Wilmington, Mass.), Taconic(Germantown, N.Y.), Harlan Sprague Dawley (Indianapolis, Ind.), etc.

The procedures for manipulation of the rodent embryo and formicroinjection of DNA into the pronucleus of the zygote are well knownto those of ordinary skill in the art (Hogan et al., supra).Microinjection procedures for fish, amphibian eggs and birds aredetailed in Houdebine and Chourrout (Experientia 47: 897-905, 1991).Other procedures for introduction of DNA into tissues of animals aredescribed in U.S. Pat. No., 4,945,050 (Sandford et al., Jul. 30, 1990).

By way of example only, to prepare a transgenic mouse, female mice areinduced to superovulate. Females are placed with males, and the matedfemales are sacrificed by CO₂ asphyxiation or cervical dislocation andembryos are recovered from excised oviducts. Surrounding cumulus cellsare removed. Pronuclear embryos are then washed and stored until thetime of injection. Randomly cycling adult female mice are paired withvasectomized males. Recipient females are mated at the same time asdonor females. Embryos then are transferred surgically. The procedurefor generating transgenic rats is similar to that of mice (Hammer etal., Cell 63: 1099-1112, 1990).

Methods for the culturing of embryonic stem (ES) cells and thesubsequent production of transgenic animals by the introduction of DNAinto ES cells using methods such as electroporation, calciumphosphate/DNA precipitation and direct injection also are well known tothose of ordinary skill in the art (Teratocarcinomas and Embryonic StemCells, A Practical Approach, E. J. Robertson, ed., IRL Press, 1987).

In cases involving random gene integration, a clone containing thesequence(s) of the invention is co-transfected with a gene encodingresistance. Alternatively, the gene encoding neomycin resistance isphysically linked to the sequence(s) of the invention. Transfection andisolation of desired clones are carried out by any one of severalmethods well known to those of ordinary skill in the art (E. J.Robertson, supra).

DNA molecules introduced into ES cells can also be integrated into thechromosome through the process of homologous recombination (Capecchi,Science 244: 1288-1292, 1989). Methods for positive selection of therecombination event (i.e., neo resistance) and dual positive-negativeselection (i.e., neo resistance and gancyclovir resistance) and thesubsequent identification of the desired clones by PCR have beendescribed by Capecchi, supra and Joyner et al. (Nature 338: 153-156,1989), the teachings of which are incorporated herein in their entiretyincluding any drawings. The final phase of the procedure is to injecttargeted ES cells into blastocysts and to transfer the blastocysts intopseudopregnant females. The resulting chimeric animals are bred and theoffspring are analyzed by Southern blotting to identify individuals thatcarry the transgene. Procedures for the production of non-rodent mammalsand other animals have been discussed by others (Houdebine andChourrout, supra; Pursel et al., Science 244: 1281-1288, 1989; and Simmset al., Bio/Technology 6: 179-183, 1988).

Thus, the invention provides transgenic, nonhuman mammals containing atransgene encoding a kinase of the invention or a gene effecting theexpression of the kinase. Such transgenic nonhuman mammals areparticularly useful as an in vivo test system for studying the effectsof introduction of a kinase, or regulating the expression of a kinase(i.e., through the introduction of additional genes, antisense nucleicacids, or ribozymes).

A “transgenic animal” is an animal having cells that contain DNA whichhas been artificially inserted into a cell, which DNA becomes part ofthe genome of the animal which develops from that cell. Preferredtransgenic animals are primates, mice, rats, cows, pigs, horses, goats,sheep, dogs and cats. The transgenic DNA may encode human STE20-relatedkinases. Native expression in an animal may be reduced by providing anamount of anti-sense RNA or DNA effective to reduce expression of thereceptor.

IX. Gene Therapy

STE20-related kinases or their genetic sequences will also be useful ingene therapy (reviewed in Miller, Nature 357: 455-460, 1992). Millerstates that advances have resulted in practical approaches to human genetherapy that have demonstrated positive initial results. The basicscience of gene therapy is described in Mulligan (Science 260: 926-931,1993).

In one preferred embodiment, an expression vector containingSTE20-related kinase coding sequence is inserted into cells, the cellsare grown in vitro and then infused in large numbers into patients. Inanother preferred embodiment, a DNA segment containing a promoter ofchoice (for example a strong promoter) is transferred into cellscontaining an endogenous gene encoding kinases of the invention in sucha manner that the promoter segment enhances expression of the endogenouskinase gene (for example, the promoter segment is transferred to thecell such that it becomes directly linked to the endogenous kinasegene).

The gene therapy may involve the use of an adenovirus containing kinasecDNA targeted to a tumor, systemic kinase increase by implantation ofengineered cells, injection with kinase-encoding virus, or injection ofnaked kinase DNA into appropriate tissues.

Target cell populations may be modified by introducing altered forms ofone or more components of the protein complexes in order to modulate theactivity of such complexes. For example, by reducing or inhibiting acomplex component activity within target cells, an abnormal signaltransduction event(s) leading to a condition may be decreased,inhibited, or reversed. Deletion or missense mutants of a component,that retain the ability to interact with other components of the proteincomplexes but cannot function in signal transduction may be used toinhibit an abnormal, deleterious signal transduction event.

Expression vectors derived from viruses such as retroviruses, vacciniavirus, adenovirus, adeno-associated virus, herpes viruses, several RNAviruses, or bovine papilloma virus, may be used for delivery ofnucleotide sequences (e.g., cDNA) encoding recombinant kinase of theinvention protein into the targeted cell population (e.g., tumor cells).Methods which are well known to those skilled in the art can be used toconstruct recombinant viral vectors containing coding sequences(Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, N.Y., 1989; Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley Interscience,N.Y., 1989). Alternatively, recombinant nucleic acid molecules encodingprotein sequences can be used as naked DNA or in a reconstituted systeme.g., liposomes or other lipid systems for delivery to target cells(e.g., Feigner et al., Nature 337: 387-8, 1989). Several other methodsfor the direct transfer of plasmid DNA into cells exist for use in humangene therapy and involve targeting the DNA to receptors on cells bycomplexing the plasmid DNA to proteins (Miller, supra).

In its simplest form, gene transfer can be performed by simply injectingminute amounts of DNA into the nucleus of a cell, through a process ofmicroinjection (Capecchi, Cell 22: 479-88, 1980). Once recombinant genesare introduced into a cell, they can be recognized by the cell's normalmechanisms for transcription and translation, and a gene product will beexpressed. Other methods have also been attempted for introducing DNAinto larger numbers of cells. These methods include: transfection,wherein DNA is precipitated with CaPO₄ and taken into cells bypinocytosis (Chen et al., Mol. Cell Biol. 7: 2745-52, 1987);electroporation, wherein cells are exposed to large voltage pulses tointroduce holes into the membrane (Chu et al., Nucleic Acids Res. 15:1311-26, 1987); lipofection/liposome fusion, wherein DNA is packagedinto lipophilic vesicles which fuse with a target cell (Felgner et al.,Proc. Natl. Acad. Sci. USA. 84: 7413-7417, 1987); and particlebombardment using DNA bound to small projectiles (Yang et al., Proc.Natl. Acad. Sci. 87: 9568-9572, 1990). Another method for introducingDNA into cells is to couple the DNA to chemically modified proteins.

It has also been shown that adenovirus proteins are capable ofdestabilizing endosomes and enhancing the uptake of DNA into cells. Theadmixture of adenovirus to solutions containing DNA complexes, or thebinding of DNA to polylysine covalently attached to adenovirus usingprotein crosslinking agents substantially improves the uptake andexpression of the recombinant gene (Curiel et al., Am. J. Respir. Cell.Mol. Biol., 6: 247-52, 1992).

As used herein “gene transfer” means the process of introducing aforeign nucleic acid molecule into a cell. Gene transfer is commonlyperformed to enable the expression of a particular product encoded bythe gene. The product may include a protein, polypeptide, anti-sense DNAor RNA, or enzymatically active RNA. Gene transfer can be performed incultured cells or by direct administration into animals. Generally genetransfer involves the process of nucleic acid contact with a target cellby non-specific or receptor mediated interactions, uptake of nucleicacid into the cell through the membrane or by endocytosis, and releaseof nucleic acid into the cytoplasm from the plasma membrane or endosome.Expression may require, in addition, movement of the nucleic acid intothe nucleus of the cell and binding to appropriate nuclear factors fortranscription.

As used herein “gene therapy” is a form of gene transfer and is includedwithin the definition of gene transfer as used herein and specificallyrefers to gene transfer to express a therapeutic product from a cell invivo or in vitro. Gene transfer can be performed ex vivo on cells whichare then transplanted into a patient, or can be performed by directadministration of the nucleic acid or nucleic acid-protein complex intothe patient.

In another preferred embodiment, a vector having nucleic acid sequencesencoding a STE20-related kinase polypeptide is provided in which thenucleic acid sequence is expressed only in specific tissue. Methods ofachieving tissue-specific gene expression are set forth in InternationalPublication No. WO 93/09236, filed Nov. 3, 1992 and published May 13,1993.

In all of the preceding vectors set forth above, a further aspect of theinvention is that the nucleic acid sequence contained in the vector mayinclude additions, deletions or modifications to some or all of thesequence of the nucleic acid, as defined above.

In another preferred embodiment, a method of gene replacement is setforth. “Gene replacement” as used herein means supplying a nucleic acidsequence which is capable of being expressed in vivo in an animal andthereby providing or augmenting the function of an endogenous gene whichis missing or defective in the animal.

X. Administration of Substances

Methods of determining the dosages of compounds to be administered to apatient and modes of administering compounds to an organism aredisclosed in U.S. application Ser. No. 08/702,282, filed Aug. 23, 1996and International patent publication number WO 96/22976, published Aug.1, 1996, both of which are incorporated herein by reference in theirentirety, including any drawings, figures, or tables. Those skilled inthe art will appreciate that such descriptions are applicable to thepresent invention and can be easily adapted to it.

The proper dosage depends on various factors such as the type of diseasebeing treated, the particular composition being used, and the size andphysiological condition of the patient. Therapeutically effective dosesfor the compounds described herein can be estimated initially from cellculture and animal models. For example, a dose can be formulated inanimal models to achieve a circulating concentration range thatinitially takes into account the IC₅₀ as determined in cell cultureassays. The animal model data can be used to more accurately determineuseful doses in humans.

Plasma half-life and biodistribution of the drug and metabolites in theplasma, tumors, and major organs can be also be determined to facilitatethe selection of drugs most appropriate to inhibit a disorder. Suchmeasurements can be carried out. For example, HPLC analysis can beperformed on the plasma of animals treated with the drug and thelocation of radiolabeled compounds can be determined using detectionmethods such as X-ray, CAT scan, and MRI. Compounds that show potentinhibitory activity in the screening assays, but have poorpharmacokinetic characteristics, can be optimized by altering thechemical structure and retesting. In this regard, compounds displayinggood pharmacokinetic characteristics can be used as a model.

Toxicity studies can also be carried out by measuring the blood cellcomposition. For example, toxicity studies can be carried out in asuitable animal model as follows: 1) the compound is administered tomice (an untreated control mouse should also be used); 2) blood samplesare periodically obtained via the tail vein from one mouse in eachtreatment group; and 3) the samples are analyzed for red and white bloodcell counts, blood cell composition, and the percent of lymphocytesversus polymorphonuclear cells. A comparison of results for each dosingregime with the controls indicates if toxicity is present.

At the termination of each toxicity study, further studies can becarried out by sacrificing the animals (preferably, in accordance withthe American Veterinary Medical Association guidelines Report of theAmerican Veterinary Medical Assoc. Panel on Euthanasia, Journal ofAmerican Veterinary Medical Assoc., 202: 229-249, 1993). Representativeanimals from each treatment group can then be examined by gross necropsyfor immediate evidence of metastasis, unusual illness, or toxicity.Gross abnormalities in tissue are noted, and tissues are examinedhistologically. Compounds causing a reduction in body weight or bloodcomponents are less preferred, as are compounds having an adverse effecton major organs. In general, the greater the adverse effect the lesspreferred the compound.

For the treatment of cancers the expected daily dose of a hydrophobicpharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250mg/day, and most preferably 1 to 50 mg/day. Drugs can be delivered lessfrequently provided plasma levels of the active moiety are sufficient tomaintain therapeutic effectiveness.

Plasma levels should reflect the potency of the drug. Generally, themore potent the compound the lower the plasma levels necessary toachieve efficacy.

EXAMPLES

The examples below are not limiting and are merely representative ofvarious aspects and features of the present invention. The examplesbelow demonstrate the isolation and characterization of theSTE20-related kinases of the invention.

Example 1 Isolation of cDNAs Encoding Mammalian STE20-Related ProteinKinases

Materials and Methods

Identification of Novel Clones

Total RNAs were isolated using the Guanidine Salts/Phenol extractionprotocol of Chomczynski and Sacchi (P. Chomczynski and N. Sacchi, Anal.Biochem. 162, 156 (1987)) from primary human tumors, normal and tumorcell lines, normal human tissues, and sorted human hematopoietic cells.These RNAs were used to generate single-stranded cDNA using theSuperscript Preamplification System (GIBCO BRL, Gaithersburg, Md.;Gerard, G F et al. (1989), FOCUS 11, 66) under conditions recommended bythe manufacturer. A typical reaction used 10 μg total RNA with 1.5 μgoligo(dT)₁₂₋₁₈ in a reaction volume of 60 μL. The product was treatedwith RNaseH and diluted to 100 μL with H₂O. For subsequent PCRamplification, 1-4 μL of this sscDNA was used in each reaction.

Degenerate oligonucleotides were synthesized on an Applied Biosystems3948 DNA synthesizer using established phosphoramidite chemistry,precipitated with ethanol and used unpurified for PCR. The sequence ofsome of the degenerate oligonucleotide primers and the amino acid motifthey encode is as follows: TRK1 5′-CTGAATTCGGNGCNTTYGGNAARGT- (SEQ IDNO:32) 3′ GAFGKV (sense) (SEQ ID NO:37) TRK45′-GCTGGATCCYTCNGGNGGCATCCA- (SEQ ID NO:33) 3′ WMPPE (antisense) (SEQ IDNO:38) ROS1 5′-GCNTTYGGNGARGTNTAYGARGG- (SEQ ID NO:34) 3′ AFGEVYEG(sense) (SEQ ID NO:39) CCK4b 5′-GCTGGATCCYTCNGGNSWCATCCA- (SEQ ID NO:35)3′ WMSPE (antisense) (SEQ ID NO:40) CCK4c 5′-GAGTTYGGNGARGTNTTYYTNGC-(SEQ ID NO:36) 3′ EFGEVYEG (sense) (SEQ ID NO:41)

These primers were derived from the sense and antisense strands ofconserved motifs within the catalytic domain of several protein kinases.Degenerate nucleotide residue designations are: N=A, C, G, or T; R=A orG; Y=C or T; H=A, C or T not G; D=A, G or T not C; S=C or G; and W=A orT.

PCR reactions were performed using degenerate primers applied tomultiple single-stranded cDNAs. The primers were added at a finalconcentration of 5 μM each to a mixture containing 10 mM Tris HCl, pH8.3, 50 mM KCl, 1.5 mM MgCl₂, 200 μM each deoxynucleoside triphosphate,0.001% gelatin, 1.5 U AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and1-4 μL cDNA. Following 3 min denaturation at 95° C., the cyclingconditions were 94° C. for 30 s, 50° C. for 1 min, and 72° C. for 1 min45 s for 35 cycles. PCR fragments migrating between 300-350 bp wereisolated from 2% agarose gels using the GeneClean Kit (Bio101), and T-Acloned into the pCRII vector (Invitrogen Corp. U.S.A.) according to themanufacturer's protocol.

Colonies were selected for mini plasmid DNA-preparations using Qiagencolumns and the plasmid DNA was sequenced using a cycle sequencingdye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City,Calif.). Sequencing reaction products were run on an ABI Prism 377 DNASequencer, and analyzed using the BLAST alignment algorithm (Altschul,S. F. et al., J. Mol. Biol. 215: 403-10).

Additional PCR strategies were employed to connect various PCR fragmentsor ESTs using exact or near exact oligonucleotide primers as detailed inthe results section for each cDNA. PCR conditions were as describedabove except the annealing temperatures were calculated for each oligopair using the formula: Tm=4(G+C)+2(A+T).

Isolation of cDNA Clones:

Human cDNA libraries were probed with PCR or EST fragments correspondingto STE20-related genes. Probes were ³²P-labeled by random priming andused at 2×10⁶ cpm/mL following standard techniques for libraryscreening. Pre-hybridization (3 h) and hybridization (overnight) wereconducted at 42° C. in 5×SSC, 5× Denhart's solution, 2.5% dextransulfate, 50 mM Na₂PO₄/NaHPO₄, pH 7.0, 50% formamide with 100 mg/mLdenatured salmon sperm DNA. Stringent washes were performed at 65° C. in0.1×SSC and 0.1% SDS. DNA sequencing was carried out on both strandsusing a cycle sequencing dye-terminator kit with AmpliTaq DNAPolymerase, FS (ABI, Foster City, Calif.). Sequencing reaction productswere run on an ABI Prism 377 DNA Sequencer.

Makegene Bioinformatics EST Assembler

The EST reports were downloaded from National Institute forBiotechnology Information. After uncompressing the files, the program‘report2est’ was scripted to extract the following information: 1) ESTnames, 2) GenBank Accession numbers, 3) GenBank gi numbers, 4) Clone Idnumbers, 5) the nucleotide sequences of the ESTs 6) the organism, 7) thelibrary name, 8) the name of the lab, and 9) the institution. The outputof ‘report2est’ is a file in FASTA format with all of the informationlisted above in the first line of each entry except the sequence, whichis listed in the second line of each entry. The resulting file isformatted for BLAST using ‘pressdb’ (available as part of the ncbi toolkit).

To build a gene or part of a gene from ESTs, the program ‘makegene’ wasdeveloped. Input to this program is a query sequence and theorganism/species for which a gene is to be built. An initial search ofthe formatted EST database described above is performed using BLAST(blastn). Any results that contain warnings, such as polyA tails orother repeat elements, are eliminated from future queries. The program‘blast_parse_reports’ was developed to extract the FASTA header linefrom the search results and the output is then filtered to extract onlyFASTA header lines for the desired species.

The initial results, having been filtered for warnings and species, gointo a loop in which searches against the database are repeated until nonew ESTs are found. The loop consists of the following steps: 1) whenpossible the names of both ends of the ESTs are extracted from thedatabase by searching using the ‘Clone Id’ field or the part of the ‘ESTname’ field before the .r or .s postscript, 2) any ESTs that have beenused as queries in previous loops are removed from the current query bythe program ‘subtract’, 3) the resulting list of ESTs is used to extractthe sequences from the database by the program batch_parse_fasta, 4)BLAST is run against the database using each sequence, 5) the outputfiles from BLAST containing warnings are removed, 6) the results arefiltered by species, and 7) the loop is reentered if there were new ESTsfound in the previous pass through the loop.

The ESTs chosen by ‘makegene’ are used as input for the program ‘mpd2cluster’ (Hide, W., Burke, J, and Davison, D. U. of Houston,unpublished) which clusters overlapping sequences. The programs ‘contig’(Kerlavage, T., TIGR, unpublished), ‘gde2mult’ and ‘gde2sing’ (Smith, S.W., et al., CABIOS 10, 671-675 (1994)), are used to make an alignmentand consensus sequence of the overlapping ESTs.

Results

cDNA Cloning and Characterization of STLK2

The human STLK2 cDNA sequence is composed of two overlapping ESTfragments, AA191319 and W16504, that were identified using aSmith-Waterman search of the EST database with STLK1 (MST3 GB:AF024636)as a query. The complete sequence of both clones was determined and usedto generate the full-length human STL2 sequence.

EST clone AA191319 contains a 1327 bp insert and an ORF of 1146 bp (382amino acids). EST clone W16504 contains a 2474 bp insert (not includingthe poly-A tail) and an ORF of 687 bp (382 amino acids).

The full-length human STLK2 cDNA (SEQ ID NO. 1) is 3268 bp long.AA191319 spans positions 1-1327 and W16504 positions 743-3216. Theoverlap between these two clones exhibits 100% sequence identity. Thehuman STLK2 cDNA constains a 1248 bp ORF flanked by a 181 bp 5′ UTR(1-181) and a 1784 bp 3′ UTR (1433-3216) that is followed by a 52nucleotide polyadenylated region. A polyadenylation signal (AATAAA) isfound at positions 3193-3198. The sequence flanking the first ATGconforms to the Kozak consensus for an initiating methionine, and isbelieved to be the translational start site for STLK2. Furthermore,human STLK2, and the related SOK-1 and MST3 proteins, conserve the aminoacid sequence immediately following this presumed initiating methionine.

Several EST fragments span the complete STLK2 sequence with AA191319 atthe 5′ end and W16504 at the 3′ end.

All searches against the public nucleic acid database (NRN) and proteindatabase (NRP) were conducted using the Smith-Waterman gap alignmentprogram ((Smith, T F and Waterman, M S (1981) J. Mol. Biol, 147,195-197).) with the PAM100 matrix and gap open and extension penaltiesof 14:1, respectively.

cDNA Cloning and Characterization of STLK3

A mammalian STLK3 clone, 135-31-19, was first identified from a PCRscreen with the degenerate oligos, TRK1 and TRK4, applied to a sscDNAgenerated from adult rat brain substantia nigra. Sequence analysis ofthe 457 bp insert indicated that it represented a novel member of theSTE20-subfamily of STKs.

A Smith-Waterman search of the EST database with the rat STLK3 fragmentand human STLK1 (MST3 GB:AF024636) as queries identified severaloverlapping ESTs spanning most of the human STLK3 cDNA sequence. AMakegene analysis generated a 3037 bp contig from approximately 44 ESTsequences. Since the 3′ ESTs were not commercially available, a pair ofprimers (5′-CACAGAAACGGTCAGATTCAC-3′(SEQ ID NO: 42) and5′-GATCAGGGTGACATCAAGGGAC-3′(SEQ ID NO: 43)) were derived from thisregion to generate PCR clone 3R21-20-6 from human fetal liver sscDNA.This clone and EST AA278967 were fully sequenced to generate thefull-length STLK2 cDNA sequence.

AA278967 is a 837 bp EST isolated by the IMAGE consortium from cDNA madefrom CD20+/IgD− germinal center B cells sorted from human tonsillarcells.

PCR clone 3R21-20-6 was isolated from human fetal sscDNA and contains a1116 bp insert, including a 1086 bp ORF encoding the 362 C-terminalamino acids of STLK3.

The full-length human STLK3 cDNA (SEQ ID NO. 2) is 3030 bp long.AA278967 spans positions 1-814 and 3R21-20-6 spans positions 464-1579.The overlap between these two clones exhibits 100% sequence identity.The remaining 1452 bp of 3′ UTR is derived from an assembly of multipleunconfirmed EST fragments.

The near full-length human STLK3 cDNA (SEQ ID NO.2) is 3030 bp long andconsists of a 1548 bp ORF flanked by a 1476 bp 3′ UTR (1550-3025) and a5 nucleotide polyadenylated region. A polyadenylation signal (AATAAA)begins at position 3004. Since the coding region is open throughout the5′ extent of this sequence, this is apparently a partial cDNA clonelacking the N-terminal start methionine. Six copies of a “GGCCCC” repeatwere observed in positions 21-67. Five independent ESTs (AA150838,AA286879, AA251679, AA252004, AA278967) showed the same repeat,suggesting that this sequence may be an integral region of the humanSTLK3 gene. Stronger evidence for this being the case is provided by thesequence of the murine orthologue of STLK3 represented by a 876 bp ESTW20737.

Multiple EST fragments span the complete STLK3 sequence with AA278967 atthe 5′ end and AA628477 and others at the 3′ end.

cDNA Cloning and Characterization of STLK4

The human STLK4 cDNA sequence is composed of two overlapping ESTfragments, AA297759 and AA100484, that were identified using aSmith-Waterman search of the EST database with STLK1 (MST3 GB:AF024636)as a query. The complete sequence of both clones was determined and usedto generate the near full-length human STLK4 sequence.

AA100484 is an IMAGE consortium cDNA clone isolated from the T-84colonic epithelium cell line. It has an insert of 3694 bp and a codingregion of 1146 bp (382 amino acids). A Smith-Waterman sequence alignmentagainst the NRN database showed this EST to be 71.4% identical to thehuman STE20-like kinase (GB:X99325).

W16504 is an IMAGE consortium clone isolated from a human fetal heartcDNA library. It has an insert length of 2474 bp (not including thepoly-A tail) and a coding region of 687 bp (229 amino acids). ASmith-Waterman sequence alignment of W16504 against the NRN databaseshowed this EST to be 69.2% identical to the human STE20-like kinase(GB:X99325).

The full-length human STLK2 cDNA (SEQ ID NO. 1) is 3268 bp long.AA191319 spans positions 1-1327, and W16504 positions 743-3216. Theoverlap between these two clones is 585 bp long with 100% sequenceidentity.

AA100484 is an IMAGE consortium cDNA clone isolated from the T-84colonic epithelium cell line. AA100484 covers the bulk of Human STLK4with its 3694 bp, which spans positions 146-3839 of SEQ ID NO:3. Asecond EST, AA297759, isolated from a Jurkat T cell cDNA library, spanspositions 1-271 of the human STLK4 contig. The two ESTs overlap over a126 bp stretch that has only one nucleotide discrepancy at position 149(G in AA297759 and T in AA100484). A T at this position was chosen forthe SEQ ID NO:3 based on sequence data generated from A100484. The 5′145 bp of human STLK4 contains three sequencing ambiguities (N's in SEQID NO:3) arising from sequence errors in the GenBank entry for AA297759.Three amino acid sequence ambiguities in the N-terminus of human STLK4are present also in SEQ ID NO:7 as a consequence of the sequenceinaccuracies from the EST entry.

The coding region of human STLK4 is 1242 bp long (2-1243), capable ofencoding a 414 amino acid polypeptide, and is followed by a 2596nucleotide 3′ UTR (1244-3839). Human STLK4 ends in a polyadenylatedstretch that has 18 adenines (3840-3857). A polyadenylation signal(AATAAA) is found between positions 3822-3827. Targeted-PCR cloningidentified one rat orthologue of human STLK4, clone 135-31-19. Inaddition, one murine orthologue of human STLK4 was recognized in the ESTdatabase as AA117483. None of these orthologues add additionalN-terminal sequence to the human STLK4.

The near full-length human STLK4 cDNA (SEQ ID NO.3) is 3857 bp long andconsists of a 1242 bp ORF flanked by a 2596 bp 3′ UTR (1244-3839) and an18 nucleotide polyadenylated region. Polyadenylation signals (AATAAA)begin at positions 2181 and 3822. Since the coding region is openthroughout the 5′ extent of this sequence, this is apparently a partialcDNA clone lacking the N-terminal start methionine. A near full-lengthmurine STLK4 cDNA is represented in the 1773 bp EST AA117438. It extendsan additional 21 nucleotides 5′ of the human STLK4 consensus, but sinceits coding region is open throughout the 5′ extent of the sequence, thisis also probably a partial cDNA clone lacking the N-terminal startmethionine.

Several EST fragments span the complete STLK3 sequence with AA297759 atthe 5′ end and AA100484 and others at the 3′ end.

cDNA Cloning and Characterization of STLK5

The human STLK5 cDNA sequence is composed of four overlapping sequences,A1418298, 2R96-13-1, 3R25-45-3 and R46685. A human STLK5 clone, F07734,was first identified using a Smith-Waterman search of the EST databasewith SPS_sc (U33057) as a query.

A1418298 is an IMAGE consortium cDNA clone with an 895 bp insert.

PCR clone 2R96-13-1 was isolated from human brain sscDNA using primers5′-CTCATCTGTACACACTTCATGG (SEQ ID NO:44) and5′-GATTCCCACACTGTAGATGTC(SEQ ID NO:45) derived from F07734. 2R96-13-1contains a 330 bp insert and an ORF of 330 bp (110 amino acids).

EST clone R46685 was identified using a Smith-Waterman search of the ESTdatabase with the C-terminus of SPS_sc (GB:U33057) as query. Sequenceanalysis of the 1047 bp insert identified this EST to contain an ORF of285 bp (95 amino acids) encoding the C-terminus of human STLK5.

PCR clone 3R25-45-3 was isolated from human fetal brain sscDNA usingprimers 5′-GGCCCTCGACTACATCCACCACAT (SEQ ID NO:46) and5′-CAACGAAACTAACACAGCATAAGG (SEQ ID NO:47) derived from 2R96-13-1 andR46685, respectively. 3R25-45-3 contains a 330 bp insert and an ORF of750 bp (250 amino acids).

The full-length human STLK5 cDNA (SEQ ID NO:96) is 2110 bp long andconsists of a 1119 bp ORF flanked by a 229 bp 5′ UTR and a 762 bp 3′UTR. The sequence flanking the first ATG conforms to the Kozak consensus(supra) for an initiating methionine, and is believed to be thetranslational start site for STLK5.

Several EST fragments span the complete STLK5 sequence with AA297059 andF07734 at the 5′ end and R46686 and F03423 and others at the 3′ end.

STLK5 displays a 100% match over a 41 bp stretch (position 2-42, SEQ IDNO. 97) to a human CpG island repeat (Z61277).

cDNA Cloning and Characterization of STLK6

Human STLK6 was first identified in the translated EST database(AA219667) as a novel serine threonine kinase.

The partial human STLK6 cDNA (SEQ ID NO:98) is 2,001 bp long andconsists of a 1,254 bp ORF flanked by a 75 bp 5′ UTR and a 673 bp 3′UTR. The sequence flanking the first ATG conforms to the Kozak consensus(Kozak, M., Nucleic Acids Res. 15, 8125-8148 (1987)) for an initiatingmethionine, and is believed to be the translational start site forSTLK6.

At the time of filing, inventors believe that STLK6 does not have anysignificant match in the nucleic acid database.

cDNA Cloning and Characterization of STLK7

Human STLK7 was first identified in the translated EST database(AA988954) as a novel serine threonine kinase. The original clone wasnot available through public sources, so a PCR fragment amplified fromthe sequence of AA988954 yielded 5R54-21-2.

The partial human STLK7 cDNA (SEQ ID NO:100) is 311 bp long and consistsof a 309 bp ORF. Since the coding region is open throughout the 5′ and3′ extent of this sequence, this appears to be a partial cDNA clonelacking the N-terminal start methionine and C-terminal stop codon.

STLK7 shares 80% sequence identity to human SPAK (AF099989) over a 167bp region and 50% nucleotide sequence identity to SLTK7 (SEQ ID NO. 101)over 391 nucleotides.

cDNA Cloning and Characterization of ZC1

The human ZC1 cDNA sequence is composed of two overlapping PCR clones,3R25-24-2 and R65-12-2.

A human ZC1 clone, 125-33-5, was first identified from a PCR screen withdegenerate oligos, TRK1 and TRK4, applied to sscDNA generated from humansmall airway epithelial cells (Clontech). Sequence analysis of the 503bp insert identified a 501 bp ORF (167 amino acids) with the potentialto encode a novel human STK related to the C. elegans ZC504.4 geneproduct.

PCR clone 3R25-24-2 was isolated from human SNB19 glioblastoma sscDNAusing primers 5′-ATGGCGAACGACTCTCCCGCGAA (SEQ ID NO:48) and5′-ACACCAAAATCAACAAGTTTCACCTC(SEQ ID NO:49) derived from the N-terminusof a murine orthologue of ZC1 (NIK, GB:U88984) and the original humanZC1 clone 125-33-5, respectively. 3R25-24-2 contains a 527 bp insert andan ORF of 519 bp (173 amino acids).

PCR clone R65-12-2 was isolated as follows: A Smith-Waterman search ofthe EST database with the C. elegans ZC504.4 gene (GB:Z50029) as a queryidentified a human EST (W81656) whose ORF is related to the C. elegansgene and terminates in an identical residue (Trp). A primer was designed3′ to this stop codon (5′-AGTTACAAGGAATTCCAAGTTCT (SEQ ID NO:50)) andused in a PCR reaction with a primer derived from the original human ZC1clone 125-33-5 (5′-ATGAAGAGGAAGAAATCAAACTG (SEQ ID NO:51)) using sscDNAfrom human SNB19 glioblastoma as a template. PCR clone R65-12-2 wasidentified and was found to contain a 3611 bp insert with a 3534 bp ORFencoding the C-terminal portion of human ZC1 (1178 amino acids).

The full-length human ZC1 cDNA (SEQ ID NO. 9) is 3798 bp long. Clone3R25-24-2 spans positions 1-527, and clone R65-12-2 spans positions188-3798. The overlap between these two clones exhibits 100% sequenceidentity. The human ZC1 contains a 3717 bp ORF (17-3723) flanked by a 6bp 5′ UTR and a 75 bp (3724-3798) 3′ UTR. No polyadenylation signal(AATAAA) or polyadenylated region are present in the 3′UTR. The sequenceflanking the first ATG conforms to the Kozak consensus for an initiatingmethionine, and is believed to be the translational start site for humanZC1.

Multiple EST fragments (W81656) match the 3′ end of the human ZC1 gene,but at the time of filing, the inventors believe that none exist inGenBank or the EST database that match its 5′ end.

cDNA Cloning and Characterization of ZC2

The human ZC2 cDNA sequence is composed of four overlapping PCR clones,G75-31-17, R65-24-6, 2R28-8-1, and R99-6-10.

A human ZC2 clone, G75-31-17, was first identified from a PCR screenwith degenerate oligos, ROS 1 (5′-GCNTTYGGNGARGTNTAYGARGG (SEQ IDNO:34)) and CCK4b (5′-GCTGGATCCYTCNGGNSWCATCCA (SEQ ID NO:35)), appliedto sscDNA generated from the human HLT383 primary non-small cell lungcancer tissue. Sequence analysis of the 492 bp insert identified a 492ORF (164 amino acids) with the potential to encode a novel human STKrelated to the C. elegans ZC504.4 gene product.

PCR clone R99-6-10 was isolated as follows: A Smith-Waterman search ofthe EST database with C. elegans ZC504.4 gene (GB:Z50029) as a queryidentified two overlapping human EST fragments (AA115844 and R51245)whose ORFs were related to the C. elegans gene and terminate in anidentical residue (Trp). A primer was designed 3′ to the stop codonfound in R51245 (5′-AGATGGACTGTACTGGGAGG (SEQ ID NO:52)) and used in aPCR reaction with a primer derived from AA115844(5′-ACTTTGTGCAGCTCTGTGGG (SEQ ID NO:53)) using human fetal brain sscDNAas a template. PCR clone R99-6-10 was identified and was found tocontain a 1095 bp insert with a 930 bp ORF encoding the C-terminalportion of human ZC2 (310 amino acids).

PCR clone R65-24-6 was isolated from human HT29 colon cancer cell linesscDNA using primers 5′-AAGGTTATGGATGTCACAGGG (SEQ ID NO:54) and5′-AGATGGACTGTACTGGGAGG (SEQ ID NO:52) derived from G75-31-17 andR51245, respectively. The 3′ primer used in this PCR reaction misprimedbetween positions 1634-1653 of this gene leading to the formation of atruncated product. R65-24-6 contains a 1593 bp insert and an ORF of 1593bp (531 amino acids).

PCR clone 2R28-8-1 was isolated from human colon cancer cell line HT29sscDNA using primers 5′-CTCACAAGGTTGCCAACAGG (SEQ ID NO:55) and5′-AGTCCCCACCAGAAGGTTTAC(SEQ ID NO:56) derived from R65-24-6 andR99-6-10, respectively. 2R28-8-1 contains a 1538 bp insert and an ORF of1536 bp (512 amino acids).

The partial human ZC2 cDNA (SEQ ID NO. 10) is 4055 bp long. CloneG75-31-17 spans positions 1-492, clone R65-24-6 spans positions 58-1650,clone 2R28-8-1 spans positions 1466-3003 and clone R99-6-10 spanspositions 2961-4055. The overlaping regions between these clones exhibit100% sequence identity except for a single guanine (G75-31-17) toadenosine (R65-24-6) mismatch at position 280 resulting in a Glu to Lyschange. Based on the presence of an acidic residue in this position inhuman ZC1 and ZC3 and C. elegans ZC504.4, the sequence encoding the Gluis probably correct. The human ZC2 gene contains a 3891 bp ORF (1-3891)flanked by 164 bp (3892-4055) 3′ UTR. No polyadenylation signal (AATAAA)or polyadenylated region is present in the 3′UTR.

Multiple EST fragments (R51245) match the 3′ end of the human ZC2 gene,but at the time of filing, the inventors believe that none exist inGenBank or the EST database that match its 5′ end.

cDNA Cloning and Characterization of ZC3

The human ZC3 cDNA sequence is composed of four overlapping PCR clones,G75-30-30, 3R33-5-3, 3R19-17-6, and R99-43-11.

A human ZC3 clone, G75-30-30, was first identified from a PCR screenwith degenerate oligos, ROS1 and CCK4b, applied to sscDNA generated froma human HLT370 primary non-small cell lung cancer tissue. Sequenceanalysis of the 492 bp insert identified a 492 ORF (164 amino acids)with the potential to encode a novel human STK related to the C. elegansZC504.4 gene product.

PCR clone R99-43-11 was isolated as follows: A Smith-Waterman search ofthe EST database with the C. elegans ZC504.4 gene (GB:Z50029) as a queryidentified a human EST (R54563) whose ORF is related to the C. elegansgene and terminates in an identical residue (Trp). A primer was designed3′ to the stop codon found in R54563 (5′-TCAGGGGTCAGAGGTCACG (SEQ IDNO:57)) and used in a PCR reaction with a primer derived from the 5′ endof R54563 (5′-CCCAAACCCTACCACAAATTC(SEQ ID NO:58)) using sscDNA fromhuman fetal brain as a template. PCR clone R99-43-11 was identified andwas found to contain a 719 bp insert with a 564 bp ORF encoding theC-terminal portion of human ZC3 (188 amino acids).

PCR clone 3R19-17-6 was isolated from human A549 lung cancer cell linesscDNA using primers 5′-CCCCCGGGAAACGATGACCA and5′-AGCCGCTGCCCCTCCTCTACTGT derived from G75-30-30 and R99-43-11,respectively. The 3′ primer used in this PCR reaction misprimed leadingto the formation of a truncated product. 3R19-17-6 contains a 1172 bpinsert and an ORF of 1170 bp (390 amino acids).

PCR clone 3R33-5-3 was isolated from human A549 lung cancer cell linesscDNA using primers 5′-ACCGCAACATCGCCACCTACTAC(SEQ ID NO:61) and5′-CTCGACGTCGTGGACCACC(SEQ ID NO:62) derived from G75-30-30 and3R19-17-6, respectively. 3R33-5-3 contains a 2465 bp insert and an ORFof 2463 bp (821 amino acids).

The full-length human ZC3 cDNA (SEQ ID NO. 11) is 4133 bp long. CloneG75-30-30 spans positions 1-483, clone 3R33-5-3 spans positions134-2598, clone 3R19-17-6 spans positions 2356-3512 and clone R99-43-11spans positions 3415-4133. The overlaps between these clones exhibit100% sequence identity. The human ZC3 gene contains a 3978 bp ORF(1-3978) flanked by a 152 bp 3′ UTR (3979-4133). No polyadenylationsignal (AATAAA) or polyadenylated region is present in the 3′UTR.

Multiple EST fragments (R54563) match the 3′end of the human ZC3 gene,but at the time of filing, the inventors believe that none exist inGenBank or the EST database that match its 5′ end.

cDNA Cloning and Characterization of ZC4

The human ZC4 cDNA sequence, represented by PCR fragment 3R25-27-1, wasfirst identified in the human genomic cosmid 82J11 (GB:Z833850)containing exon sequences that displayed strong homology to the ZC504.4C. elegans gene.

PCR clone 3R25-27-1 was isolated from human fetal liver sscDNA andprimers 5′-CAATGTTAACCCACTCTATGTCTC(SEQ ID NO:63) and5′-AGTTTGCCGATGTTTTTCTTTTC(SEQ ID NO:64) derived from a potential ORF(positions 25729-25852) from the 82J11 cosmid and from an EST (R98571)encoding the C-terminus of the human ZC4 gene, respectively.

The partial human ZC4 cDNA (SEQ ID NO.12) is 1459 bp long and consistsof a 1047 bp ORF (2-1048) flanked by a 411 bp (1049-1459) 3′UTR region.No polyadenylation signal (AATAAA) or polyadenylated region is presentin the 3′UTR.

The N-terminal coding sequence for ZC4_h was extended by building acontiguous DNA sequence of 233,137 bp containing Z83850 and four othersequences: cU84B10 and cU230B10 (from the Sanger Human Genome SequencingProject) and Z97356 and Z69734 (available from the National Institutefor Biotechnology Information. The position of each sequence in thecontig is represented in the table below. Accession Length Start EndcU84B10 43273 0 43273 Z97356 21848 43171 65018 Z69734 37077 63073 100149cU230B10 11841 88416 100256 Z83850 132981 100156 233137

Sequences in ZC4 genomic contig.

The 233,137 bp contig was analyzed for exons using the programs FGENES1.5 and FGENESH, human gene structure prediction software available fromThe Sanger Centre.

The resulting human ZC4 coding sequence (SEQ ID NO:104) is 3,681 bp long(excluding the stop codon) and encodes for a STE20 kinase of 1227 aminoacids.

cDNA Cloning and Characterization of KHS2

The human KHS2 cDNA sequence is composed of four overlapping clones,3R25-51-2, 3R16-34-2, 3R16-31-2, and T79916.

A human KHS2 clone, AA250855, was first identified using aSmith-Waterman search of the EST database with KHS 1 (GB:U77129) as aquery. Sequence analysis of the 1112 bp insert identified a 618 bp ORF(206 amino acids) with the potential to encode a novel STK related tothe human KHS1 gene product. Using AA250855 as a query, a second EST(AA446022) was found whose sequence was shown to contain the initiatormethionine for human KHS2 based on a comparison with KHS 1.

PCR clone 3R25-51-2 was isolated from human testicular cancer sscDNAusing primers 5′-CCGCCATGAACCCCGGCTT (SEQ ID NO:65) and5′-CGATTGCCAAAGACCGTGTCA (SEQ ID NO:66) derived from AA446022 andAA250855, respectively. 3R25-51-2 contains an 850 bp insert and an ORFof 849 bp (283 amino acids).

EST clone, T79916, was identified using a Smith-Waterman search of theEST database with the C-terminus of KHS1 (GB:U77129) as a query.Sequence analysis of the 2107 bp insert identified this EST to containan ORF of 345 bp (115 amino acids disrupted by a single stop codon)encoding the C-terminus of human KHS2, followed by 1762 bp 3′UTR.

PCR clone 3R16-34-2 was isolated from human testis sscDNA using primers5′-AGAAGTTGCAGCTGTTGAGAGGA (SEQ ID NO:67) and5′-TATGGCCCGTGTAAGGATTTC(SEQ ID NO:68) derived from AA250885 and T79916,respectively. 3R16-34-2 contains an 1516 bp insert and an ORF of 1128 bp(376 amino acids).

PCR clone 3R16-31-2 was isolated from normal human colon sscDNA usingprimers 5′-GTGCCAGAAGTGTTGTGTTGTAA (SEQ ID NO:69) and5′-TATGGCCCGTGTAAGGATTTC(SEQ ID NO:68) derived from EST T79916.3R16-31-2 contains a 728 bp insert and an ORF of 669 bp (223 aminoacids). This clone lacked the stop codon present within EST T79916(postion 2662 in the KHS2 sequence).

The full-length human KHS2 cDNA (SEQ ID NO.17) is 4023 bp long. Clone3R25-51-2 spans positions 1-855, clone AA250885 spans positions 336-923,clone 3R16-34-2 spans positions 545-2061, and clone T79916 spanspositions 1917-4023. The overlaping regions between these clones exhibit100% sequence identity, except for 4 nucleotide differences, two ofwhich are silent, a third corrects the internal stop codon at position2662, and the fourth at position 247 (T to C change) results in a Pro toLeu change. The human KHS2 cDNA contains a 2682 bp ORF (6-2687) flankedby a 5 bp (1-5) 5′UTR and a 1336 bp (2688-4023) 3′ UTR. A potentialpolyadenylation signal (AATAAA) is found at positions 4008-4013. Nopolyadenylated region is present in the 3′UTR. The sequence flanking thefirst ATG is in a poor context for translational initiation, however, a134 bp 5′UTR sequence from EST AA446022 did not reveal any additionalATG's and displayed two in-frame stop codons 5′ to the putative startATG for human KHS2.

Multiple EST fragments match the 5′end (AA446022) as well as the 3′ end(R37625) of the human KHS2 gene.

cDNA Cloning and Characterization of SULU1

The human SULU1 cDNA sequence is composed of three overlapping clones,N40091, 2R90-1-1 and R90907.

A human SULU1 clone, N40091, was first identified using a Smith-Watermansearch of the EST database with the C. elegans SULU gene (GB: U32275) asa query. Sequence analysis of the 1321 bp insert identified a 906 bp ORF(302 amino acids) with the potential to encode a novel human STK relatedto the C. elegans SULU gene product.

EST clone R90907 was first identified using a Smith-Waterman search ofthe EST database with the 3′ end of the C. elegans SULU gene (GB:U32275) as a query. Sequence analysis of the 1647 bp insert identified a578 bp ORF (192 amino acids) with the potential to encode the C-terminusof the human SULU1 gene product.

PCR clone 2R90-1-1 was isolated from human HT29 colon cancer cell sscDNAusing primers 5′-TATTGAATTGGCGGAACGGAAG (SEQ ID NO:70) and5′-TTGTTTTGTGCTCATTCTTTGGAG (SEQ ID NO:71) derived from N40091 andR90907, respectively. 2R90-1-1 contains a 1625 bp insert and an ORF of1623 bp (541 amino acids).

The full-length human SULU1 cDNA (SEQ ID NO.19) is 4177 bp long CloneN40091 spans positions 1-1321, clone 2R90-1-1 spans positions 1048-2671,and clone R90907 spans positions 2531-4177. The overlaping regionsbetween these clones exhibit 100% sequence identity. The human SULU1cDNA contains a 2694 bp ORF (415-3108) flanked by a 414 bp (1-414) 5′UTRand a 1069 bp (3109-4177) 3′ UTR followed by a 19 nucleotidepolydenylated region. A potential polyadenylation signal (AATAAA) isfound at positions 4164-4169. The sequence flanking the first ATGconforms to the Kozak consensus for an initiating methionine, and isbelieved to be the translational start site for human SULU1.

Multiple EST fragments match the 5′end (N27153) as well as the 3′ end(R90908) of the human SULU1 gene.

cDNA Cloning and Characterization of Murine SULU3

The murine SULU3 cDNA sequence is represented by PCR fragment 2R92-1-6.

A murine SULU3 clone, G83-4-5, was first identified from a PCR screenwith degenerate oligos, CCK4c and CCK4b, applied to sscDNA generatedfrom murine day-12 embryos. Sequence analysis of the 473 bp insertidentified a 471 ORF (157 amino acids) with the potential to encode anovel human STK related to the C. elegans SULU gene (GB: U32275)product. The antisense strand of G83-4-5 is identical at the nucleicacid level to the 5′UTR of the murine ets1 protooncogenic transcriptionfactor (GB:X53953). This homology is likely the result of a cloningartifact attached to the 5′-end of the database entry for murine ets1.

PCR clone 3R19-17-6 was isolated from human A549 cell sscDNA usingprimers 5′-CCCCCGGGAAACGATGACCA (SEQ ID NP:59) and5′-AGCCGCTGCCCCTCCTCTACTGT (SEQ ID NO:60) derived from G75-30-30 andR99-43-11, respectively. The 3′ primer used in this PCR reactionmisprimed leading to the formation of a truncated product. 3R19-17-6contains a 1172 bp insert and an ORF of 1170 bp (390 amino acids).

PCR clone 2R92-1-6 was isolated from murine d8 embryo sscDNA usingprimers 5′-ACCGCAACATCGCCACCTACTAC(SEQ ID NO:61) and5′-GATTGCTTTGTGCTCATTCTTTGG (SEQ ID NO:72) derived from the 5′ UTR ofthe ets1 gene and the human EST AA234623, respectively. The latter(shown herein) encodes the C-terminus of human SULU3. 2R92-1-6 containsa 2249 bp insert and an ORF of 2244 bp (748 amino acids).

The partial murine SULU3 cDNA (SEQ ID NO.21) is 2249 bp long andconsists of a 2244 bp ORF (6-2249) flanked by a 5 bp (1-5) 5′UTR. Thesequence flanking the first ATG conforms to the Kozak consensus for aninitiating methionine, and is believed to be the translational startsite for murine SULU3.

One EST fragment (AA446022) matches the 3′ end of the partial murineSULU3 gene, but at the time of filing, the inventors believe that noneexist in GenBank or the EST database that match its 5′ end.

cDNA Cloning and Characterization of Human SULU3

The human SULU3 cDNA sequence is composed of two overlapping clones,2R90-22-1 and AA234623.

A human SULU3 clone, AA234623, was first identified using aSmith-Waterman search of the EST database with the C. elegans SULU gene(GB: U32275) as a query. Sequence analysis of the 2652 bp insertidentified a 1185 bp ORF (395 amino acids) with the potential to encodethe C-terminus of a novel human STK related to the C. elegans SULU geneproduct.

PCR clone 2R90-22-1 was isolated from human SKMel128 melanoma cell linesscDNA using primers 5′-TATTGAATTGGCGGAACGGAAG (SEQ ID NO:70) and5′-TTGTTCTAAGAGTGCCCTCCG (SEQ ID NO:73) derived from the murine SULU32R92-1-6 clone and from AA234623, respectively. 2R92-1-6 contains a 1897bp insert and an ORF of 1896 bp (632 amino acids).

The partial human SULU3 cDNA (SEQ ID NO.20) is 3824 bp long. Clone2R90-22-1 spans positions 1-1897 and clone AA234623 spans positions1173. The overlaping region between these clones exhibits 100% sequenceidentity. The human SULU3 cDNA contains a 2358 bp ORF (2-2359) flankedby a 1465 bp (2360-3824) 3′UTR followed by a 19 nucleotide polydenylatedregion. A potential polyadenylation signal (AATAAA) is found atpositions 2602-2607. Since the coding region is open throughout the 5′extent of this sequence, this is apparently a partial cDNA clone lackingthe N-terminal start methionine.

Multiple EST fragments (R02283) match the 3′end of the human SULU3 gene,but at the time of filing, the inventors believe that none exist inGenBank or the EST database that match its 5′ end.

cDNA Cloning and Characterization of GEK2

The human GEK2 cDNA sequence is composed of three overlapping clones,AA459448, 3R25-48-1 and GEK2_h#3.

A human GEK2 clone, AA459448, was first identified using aSmith-Waterman search of the EST database with the human SLK gene (GB:AB002804) as a query. Sequence analysis of the 1286 bp insert identifieda 1227 bp ORF (409 amino acids) with the potential to encode theN-terminus of a novel human STK related to the human SLK gene product.An additional Smith-Waterman search using the C-terminus of the SLK geneas a query yielded three additional EST's, AA323687, AA380492 andAA168869, that encode the C-terminal region of human GEK2.

PCR clone 2R98-41-17 was isolated from human testis sscDNA using primers5′-AAGACCATGCCGTGCGCCG (SEQ ID NO:74) and 5′-ATTCCTTCAGGTTCTGGTTATGG(SEQ ID NO:75) derived from AA323687 and from AA380492, respectively.2R98-41-17 contains a 851 bp insert and an ORF of 849 bp (283 aminoacids).

PCR clone GEK2_h#3 was isolated from human sscDNA made from the H23tumor cell line using primers 5′-GCAGCAAGTGGAGAAGATGG (SEQ ID NO:109)and 5′-GGAAGCATCCCCAGAGCTGTAG (SEQ ID NO:110) derived from the sequenceof clone 3R25-48-1 and from the 3′ end of murine LOK (GB:D89728),respectively. GEK2_h#3 contains a 1042 bp insert and an ORF of 1041 bp(347 amino acids).

The full-length human GEK2 cDNA (SEQ ID NO:106) is 2962 bp long. CloneAA459448 spans positions 1-1286, clone 3R25-48-1 spans positions1100-2449 and clone GEK2_h#3 spans positions 1920-2962. The overlappingregions between these clones exhibit 100% sequence identity.

The human GEK2 cDNA contains a 2904 bp ORF (59-2962) flanked by a 58 bp(1-58) 5′UTR. The sequence flanking the first ATG conforms to the Kozakconsensus for an initiating methionine, and is believed to be thetranslational start site for human GEK2.

Multiple EST fragments (AA465671) match the 5′end of the sequence, butonly one (AA380492) matches the 3′ end of the human GEK2 gene.

cDNA Cloning and Characterization of PAK4

The human PAK4 cDNA sequence is represented by clone SNB2#1.

A human PAK4 clone, R88460, was first identified using a Smith-Watermansearch of the EST database with the human PAK gene (GB: U24152) as aquery. Sequence analysis of the 2332 bp insert identified a 930 bp ORF(310 amino acids) with the potential to encode the C-terminus of a novelhuman STK related to the human PAK gene product.

cDNA clone SNB2#1 was isolated from human glioblastoma cell line SNB75cDNA library using a probe derived from R88460. SNB2#1 contains a 3604bp insert and an ORF of 2043 bp (681 amino acids).

The full-length human PAK4 cDNA (SEQ ID NO.27) is 3604 bp long andconsists of a 2043 bp ORF (143-2185) flanked by a 142 bp (1-142) 5′UTRand a 1419 3′ UTR followed by a 22 nucleotide polydenylated region. Apotential polyadenylation signal (AATTAAA) is found at positions3582-3588. The sequence flanking the first ATG conforms to the Kozakconsensus for an initiating methionine, and is believed to be thetranslational start site for human PAK4. The 3′ UTR of the PAK4 genecontains a GT dinucleotide repeat prone to undergo expansion based onthe number of repeats found in clones SNB#1 and R88460, 32 and 23,respectively. Several neurologic disorders have been correlated with theexpansion of di- or tri-nucleotide repeats similar to those found in thePAK4 sequence, suggesting PAK 4 may also be a disease target and thatthis repeat in its 3′UTR may serve as a diagnostic marker.

Multiple EST fragments (AA535791) match the 3′end of the human PAK4gene, but at the time of filing, the inventors believe that none existin GenBank or the EST database that match its 5′ end.

cDNA Cloning and Characterization of PAK5

The full-length human PAK5 cDNA sequence is composed of two overlappingclones, H450#1-1 and SNB8#5.

A human PAK5 clone, R18825, was first identified using a Smith-Watermansearch of the EST database with the human PAK4 gene as a query. Sequenceanalysis of the 1248 bp insert identified a 420 bp ORF (140 amino acids)with the potential to encode the C-terminus of a novel human STK relatedto the human PAK4 gene product.

cDNA clone SNB8#5 was isolated from human SNB75 cDNA library using aprobe derived from R18825. SNB2#1 contains a 2028 bp insert and an ORFof 1194 bp (398 amino acids).

The partial human PAK5 cDNA (SEQ ID NO.28) is 2028 bp long and consistsof a 1194 bp ORF (2-1195) flanked by an 833 bp (1196-2028) 3′UTRfollowed by a 22 nucleotide polydenylated region. A potentialpolyadenylation signal (AATTAAA) is found at positions 2004-2010. Sincethe coding region is open throughout the 5′ extent of this sequence,this is apparently a partial cDNA clone lacking the N-terminal startmethionine.

Clone H460#1-1 was isolated from a human lung H460 cDNA library using aprobe derived from the partial SNB2#1 cDNA clone described above.Sequence analysis of the 2526 bp insert identified a 1773 bp ORF (592amino acids) with the potential to encode a full-length PAK5.

The human PAK5 cDNA (SEQ ID NO:102) is 2,806 bp long and consists of a1,773 bp ORF flanked by a 201 bp 5′ UTR and a 833 bp 3′ UTR. Thesequence flanking the first ATG conforms to the Kozak consensus (Kozak,M., Nucleic Acids Res. 15, 8125-8148 (1987)) for an initiatingmethionine, and is believed to be the translational start site for PAK5.

PAK5 shares 99% sequence identity over 2795 bp to a recent databaseentry, AF005046. These sequences are presumed to be from the same gene,with minor polymorphic variations.

Example 2 Expression Analysis of Mammalian STE20-related Protein Kinases

Materials and Methods

Northern Blot Analysis

Northern blots were prepared by running 10 μg total RNA isolated from 60human tumor cell lines (HOP-92, EKVX, NCII-H23, NCI-H226, NCI-H322M,NCI-H460, NCI-H522, A549, HOP-62, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8,IGROV1, SK− OV-3, SNB-19, SNB-75, U251, SF-268, SF-295, SF-539,CCRF-CEM, K-562, MOLT-4, HL-60, RPMI 8226, SR, DU-145, PC-3, HT-29,HCC-2998, HCT-116, SW620, Colo 205, HTC15, KM-12, UO-31, SN12C, A498,CaKil, RXF-393, ACHN, 786-0, TK-10, LOX IMVI, Malme-3M, SK-MEL-2,SK-MEL-5, SK-MEL-28, UACC-62, UACC-257, M14, MCF-7, MCF-7/ADR RES,Hs578T, MDA-MB-231, MDA-MB-435, MDA-N, BT-549, T47D), from 22 humanadult tissues (thymus, lung, duodenum, colon, testis, brain, cerebellum,cortex, salivary gland, liver, pancreas, kidney, spleen, stomach,uterus, prostate, skeletal muscle, placenta, mammary gland, bladder,lymph node, adipose tissue), and 2 human fetal normal tissues (fetalliver, fetal brain), on a denaturing formaldehyde 1.2% agarose gel andtransferring to nylon membranes.

Filters were hybridized with random primed [α³²P]dCTP-labeled probessynthesized from the inserts of several of the STE20-related kinasegenes. Hybridization was performed at 42° C. overnight in 6×SSC, 0.1%SDS, 1× Denhardt's solution, 100 μg/mL denatured herring sperm DNA with1-2×10⁶ cpm/mL of ³²P-labeled DNA probes. The filters were washed in0.1×SSC/0.1% SDS, 65° C., and exposed on a Molecular Dynamicsphosphorimager.

Quantitative PCR Analysis

RNA was isolated from a variety of normal human tissues and cell lines.Single stranded cDNA was synthesized from 10 □g of each RNA as describedabove using the Superscript Preamplification System (GibcoBRL). Thesesingle strand templates were then used in a 25 cycle PCR reaction withprimers specific to each clone. Reaction products were electrophoresedon 2% agarose gels, stained with ethidium bromide and photographed on aUV light box. The relative intensity of the STK-specific bands wereestimated for each sample.

DNA Array Based Expression Analysis

Plasmid DNA array blots were prepared by loading 0.5 □g denaturedplasmid for each STE20-related kinase on a nylon membrane. The[α³²P]dCTP labeled single stranded DNA probes were synthesized from thetotal RNA isolated from several human immune tissue sources or tumorcells (thymus, dendrocytes, mast cells, monocytes, B cells (primary,Jurkat, RPMI8226, SR), T cells (CD8/CD4+, TH1, TH2, CEM, MOLT4), K562(megakaryocytes). Hybridization was performed at 42° C. for 16 hours in6×SSC, 0.1% SDS, 1× Denhardt's solution, 100 μg/mL denatured herringsperm DNA with 106 cpm/mL of [α³²P]dCTP labeled single stranded probe.The filters were washed in 0.1×SSC/0.1% SDS, 65° C., and exposed forquantitative analysis on a Molecular Dynamics phosphorimager.

Results

Distribution of STE20-Related Gene Transcripts in Normal Tissues andTumor Cell Lines

ZC1, ZC2, and ZC3 RNA expression was analyzed by quantitative PCR frommultiple human normal tissues, cultured primary epithelial andendothelial cells, and tumor cell lines. The results are summarized inTables 1 and 2, with relative expression values ranging from 0(undetectable) to 23 (very strong). An “x” refers to sample not tested.ZC1, ZC2, and ZC3 were all expressed at very low levels in most normalhuman tissues, however ZC1 and ZC2 were more abundant in culturedepithelial cells and ZC3 in normal kidney and breast tissue.

Expression of these 3 genes was also examined in a panel of human tumorcell lines representing a diverse sampling of tumor types (Table 2). ZC1and ZC2 showed strong expression in cell lines from most melanomas andrenal tumors and from some non-small cell lung cancers and colon tumors.ZC3 expression was consistently lower in the tumor cell lines except forhigh expression in most breast cancers and leukemias. The robustoverexpression ZC1, ZC2, and ZC3 in tumor cells versus normal tissuesmay provide an attractive target for oncology drug development.

Expression of all the novel STE20-related kinases was examined in apanel of human immune tissues/cells by hybridization to a DNA array blotcontaining plasmids encoding each of these genes. STLK2 was broadlyexpressed in all 14 immune samples, whereas STLK4 and PAK4 were highlyexpressed in a subset of 6-7 of the samples (Table 3). Several otherkinases (SULU3, ZC4, KHS2) had more restricted expression, while otherswere expressed in only a single immune source (STLK3, thymus; ZC1,dendrocytes; ZC3, monocytes; PAK5, mast cells and MOLT4), and severalmore were absent from all the immune sources assayed (GEK2, SULU1, ZC2,STLK5). These expression patterns were quite distinct among members ofthe same subfamily (i.e., ZC1, ZC2, ZC3 and ZC4, or PAK1, PAK2, PAK3,PAK4, PAK5). This analysis suggests that some of these kinases may becandidate targets for various immune disorders, and that some, which aremore broadly expressed, may mediate functions vital to the basic biologyof most proliferating cells. TABLE 1 ZC1, ZC2 and ZC3 Expression inNormal Human Tissues and Cells Sample ZC1 ZC2 ZC3 NORMAL Brain Tiss 2.80.6 0.9 Duod Tiss 3.8 1.5 0.3 Heart Tiss 1.2 0.3 0.0 Kidney Tiss 0.7 0.07.0 Lung Tiss 1.6 0.2 0.0 Pancreas Tiss 2.0 0.4 2.5 Placenta Tiss 1.40.0 0.0 Sal gl. Tiss 3.0 0.3 3.2 Sk mus. Tiss 2.3 0.1 0.1 Spleen Tiss0.4 0.0 x Stomach Tiss 0.8 0.0 0.0 Thymus Tiss 3.5 0.4 1.5 Cereb Tiss2.8 1.1 4.4 Liver Tiss 1.8 0.0 0.4 Uterus Tiss 1.6 0.0 1.4 Prostate Tiss1.4 0.0 1.6 Testis Tiss x x 5.8 f Brain Tiss x x 3.1 Mam gl Tiss x x 7.2HCAEC ENDO 1.0 0.0 0.0 HMVEC-d ENDO 0.7 0.0 0.4 HMVEC-L ENDO 2.2 1.6 1.8HPAEC ENDO 9.3 5.3 6.4 HMEC EPI 4.1 2.3 1.9 RPTEC EPI 3.6 2.2 0.2 HRCEEPI 5.3 3.5 1.3 HSAE EPI 0.9 3.3 4.8

TABLE 2 ZC1, ZC2 and ZC3 Expression in Tumor Cell lLnes Sample OriginZC1 ZC2 ZC3 HOP-92 Lung 9.3 7.2 3.3 EKVX Lung 10.7 3.7 3.5 NCI-H23 Lung5.8 6.3 4.1 NCI-H226 Lung 6.5 6.8 3.3 NCI-H322M Lung 3.5 5.8 4.9NCI-H460 Lung 4.5 3.7 2.9 NCI-H522 Lung 4.7 3.3 4.6 A549/ATCC Lung 3.83.6 4.1 HOP-62 Lung 4.3 3.8 4.2 OVCAR-3 Ovary 2.9 3.1 1.5 OVCAR-4 Ovary3.3 1.0 3.8 OVCAR-5 Ovary 2.6 3.6 2.2 OVCAR-8 Ovary 3.6 2.0 4.7 IGROV1Ovary 3.8 1.7 3.2 SK-OV-3 Ovary 4.9 0.0 3.5 SNB-19 CNS 5.1 5.4 4.2SNB-75 CNS 2.5 0.9 0.7 U251 CNS 1.5 1.2 0.6 SF-268 CNS 5.8 2.7 3.0SF-295 CNS 6.4 1.1 3.2 SF-539 CNS 5.1 2.9 4.3 CCRF-CEM Leuk 3.4 2.7 3.1K-562 Leuk 4.1 6.3 4.3 MOLT-4 Leuk 7.1 3.4 4.2 HL-60 Leuk x x 0.4 RPMI8226 Leuk 0.5 0.2 1.4 SR Leuk 3.5 7.2 5.4 DU-145 Pro x x 3.4 PC-3 Pro xx 3.4 HT-29 Colon 2.4 5.9 6.6 HCC-2998 Colon 2.4 3.8 3.0 HCT 116 Colon2.2 2.1 5.4 SW-620 Colon 7.8 12.1 3.1 COLO 205 Colon 9.1 16.2 3.0 HCT-15Colon 13.8 4.9 2.5 KM-12 Colon 7.0 13.2 3.1 UO-31 Colon 10.4 10.6 0.9SN12C Renal 8.1 3.4 2.8 A498 Renal 6.2 3.1 2.9 Caki-1 Renal 9.2 14.4 2.3RXF 393 Renal 10.6 4.8 2.8 ACHN Renal 9.3 6.0 3.9 786-0 Renal 8.8 15.65.6 TK-10 Renal 20.9 21.2 5.0 LOX IMVI Mel 2.3 2.4 3.3 Malme-3M Mel x x2.2 SK-MEL-2 Mel 15.7 14.1 2.9 SK-MEL-5 Mel 7.9 7.0 0.0 SK-MEL-28 Mel16.5 23.1 0.0 UACC-62 Mel 12.1 18.3 5.3 UACC-257 Mel 10.8 9.4 6.2 M14Mel 4.4 0.9 7.9 MCF7 Breast 4.8 1.3 7.7 MCF-7/ADR Breast 8.8 3.4 7.7 Hs578T Breast 6.9 2.6 5.7 MDA-MB-231 Breast 5.7 1.9 6.4 MDA-MB-435 Breast4.8 6.7 9.1 MDA-N Breast 7.3 6.3 9.1 BT-549 Breast 3.6 1.9 8.0 T-47DBreast 0.4 12.3 9.3

TABLE 3 STE20-related kinase expression in a human immune panel MastCD8+ KINASE thymus Dendrocytes cells Monocytes B cells CD4+ TH1 TH2 GEK2350 350 350 350 350 350 350 350 SULU1 350 350 350 350 350 350 350 350SULU3 350 350 350 350 12149 350 5115 350 STLK2 117770 13771 27620 9203618305 39109 5408 3564 STLK3 8624 350 350 350 350 350 350 350 STLK4 8524350 350 350 350 8685 5642 350 STLK5 xxx xxx xxx xxx 350 350 350 xxx ZC1350 3377 350 350 350 350 350 350 ZC2 350 350 350 350 350 350 350 350 ZC3350 350 350 20156 350 350 350 350 ZC4 xxx xxx xxx xxx 350 350 350 xxxKHS2 8766 2508 350 56575 350 350 350 350 PAK4 32658 7684 3729 100948 350350 350 1604 PAK5 350 350 4905 350 350 350 350 350 CEM MOLT4 JURKATRPMI8226 SR K562 KINASE (T cell) (T cell) (B cell) (B cell) (B cell)(MO) GEK2 350 350 350 350 350 350 SULU1 350 350 350 350 350 350 SULU3350 350 350 350 350 350 STLK2 47236 53262 47605 22560 65936 30390 STLK3350 350 350 350 350 350 STLK4 3648 350 26772 1570 350 350 STLK5 350 350350 xxx 350 350 ZC1 350 350 350 350 350 350 ZC2 350 350 350 350 350 350ZC3 350 350 350 350 350 350 ZC4 1094 7813 14945 xxx 350 6385 KHS2 350350 350 350 350 350 PAK4 350 10246 350 3229 350 350 PAK5 350 12672 350350 350 350

Transcript size from Northern data Kinase (kb) STLK2 3.8 STLK4 5.0 ZC16.9/4.7 ZC2 6.0/8.0 ZC4 5 KHS2 4.4 SULU1 4.5 SULU3 10.0 GEK2 5.5 PAK44.8 PAK5 3.5

STLK2 is widely expressed; the highest expression levels were found inplacenta, spleen and PBL.

STLK4 is also widely expressed in normal tissues including heart, brain,placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,thymus, prostate, testis, ovary, small intestine, colon, and peripheralblood lymphocytes. STLK4 was also detected in Jurkat T cells.

ZC1 is highly overexpressed in the following human cancer cell lines:HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H522, A549, HOP-62(lung); OVCAR-3, OVCAR-4, OVCAR-5 (ovary); SNB-19, U251, SF-268, SF-295,SF-539 (CNS); K-562, RPMI-8226 (leukemia); DU-145, PC-3 (prostate);HT-29, HCC-2998, HCT-116, SW620, COLO-205, HCT-15, KM-12 (colon); UO-31,CAKi-1, RXF-393, 786-0, TK-10 (renal); LOXIMVI, Malme-3M, SK-MEL-2,SK-MEL-28, UACC-62, UACC-257, M14 (melanoma); and MCF-7, MCF-7/ADR, HIS578T, MDA-MB-231, MDA-MB-431, MDA-N, BT-549, T-47D (breast).

ZC2 is expressed in brain and testis. It is highly overexpressed in thefollowing human cancer cell lines: TK-10 (renal); SK-MEL-28, UACC-62(melanoma); T47D (breast).

Moderate expression in HOP92 (lung); OVCAR4, IGROV1 (ovary); DNB75, U251(brain); K-562 (leukemia); and COL0205 (colon).

SULU1 is overexpressed in the following human cancer cell lines: HOP-92,EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H522, A549, HOP-62 (lung);OVCAR-3, OVCAR-4, OVCAR-5, SK-OV-3 (ovary); SNB-19, U251, SF-268,SF-295, SF-539 (CNS); K-562, RPMI-8226 (leukemia); DU-145, PC-3(prostate); HT-29, HCC-2998, HCT-116, SW620, COLO-205, HCT-15, KM-12(colon); UO-31, CAKi-1, RXF-393, 786-0, TK-10 (renal); LOX, IMVI,Malme-3M, SK-MEL-2, SK-MEL-28, UACC-62, UACC-257, M14 (melanoma); MCF-7,MCF-7/ADR, HIS 578T, MDA-MB-231, MDA-MB-431, MDA-N, BT-549, T-47D(breast).

SULU3 showed a broad pattern of expression in the normal tissue panel ofRNAs.

GEK2 was expressed in spleen, thymus and testis. Expression was high inthe cell lines RBL-2H3 and H441.

PAK4 was expressed in the normal tissues: brain, testis and prostate,and in the human cancer cell lines: HNCI-H23 (lung); OVCAR-3 (ovary);SNB-19, U251 (CNS); RPMI-8226 (leukemia); DU-145 (prostate); COLO-205,HCT-15 (colon).

PAK5 showed weak expression levels in the normal tissues: brain, testes,bladder, colon, adrenal medulla, spleen, fetal liver, breast, cerebralcortex, cerebellum, thymus, salivary gland, lung, stomach, duodenum,uterus, prostate, skeletal muscle and placenta. PAK5 was overexpressedin the human cancer cell lines: HOP-92, EKVX, NCI-H23, NCI-H226,NCI-H322M, NCI-H522, A549, HOP-62 (lung); OVCAR-3, OVCAR-4, OVCAR-5,SK-OV-3 (ovary); SNB-19, U251, SF-268, SF-295, SF-539 (CNS); K-562,RPMI-8226 (leukemia); DU-145, PC-3 (prostate); HT-29, HCC-2998, HCT-116,SW620, COLO-205, HCT-15, KM-12 (colon); UO-31, CAKi-1, RXF-393, 786-0,TK-10 (renal); LOXIMVI, Malme-3M, SK-MEL-2, SK-MEL-28, UACC-62,UACC-257, M14 (melanoma); MCF-7, MCF-7/ADR, HIS 578T, MDA-MB-231,MDA-MB-431, MDA-N, BT-549, T-47D (breast).

Example 3 STE20-Related Protein Kinase Gene Expression VectorConstruction

Materials and Methods

Expression Vector Construction

Several expression constructs were generated for some of the humanSTE20-related cDNAs including: a) full-length clones in a pCDNAexpression vector; b) a GST-fusion construct containing the catalyticdomain of the novel STE20-related kinase fused to the C-terminal end ofa GST expression cassette; and c) a full-length clone containing a Lysto Ala (K to A) mutation at the predicted ATP binding site within thekinase domain, inserted in the pCDNA vector.

The “K to A” mutants of the STE20-related kinase might function asdominant negative constructs, and will be used to elucidate the functionof these novel STKs.

Results

Constructs for ZC1, ZC2, ZC3, SULU1, SULU3, PAK4 and PAK5 have beengenerated.

Numerous additional constructs have been generated for the variousSTE20-subfamily kinases, including full length, kinase inactive andtagged versions. In addition, the following three constructs weredesigned for specific applications based on their unique domainstructure:

-   Construct 1: SULU1-coiled-coil2-   Vector: pGEX-4T-   Insert: Coiled-coil2-   Sequence: Amino acids 752-898-   Purpose: phage display-   Result: Interacts with GEK2 CC1-   Construct 2: SULU3-coiled-coil2-   Vector: pGEX4T-   Insert: coiled-coil 2 domain fused to GST-   Sequence range of insert: amino acids 802-898 of SEQ-   Purpose: phage display-   Result: Interacts with coiled-coiled region of human SLK-   Construct 3: PAK5 Dominant Negative-   Vector: pCAN5-   Insert: Full length coding sequence of human PAK5 containing the    following mutation: K350,351A (Lys at aa positions 350 and 351    changed to Ala).-   Purpose: to determine role of human PAK5 kinase activity in cell    growth and transformation.-   Result: Interferes with Ras transformation.

Example 4 Generation of Specific Immunoreagents to STE20-Related ProteinKinases

Materials and Methods

Specific immunoreagents were raised in rabbits against KLH- orMAP-conjugated synthetic peptides corresponding to the humanSTE20-related kinases. C-terminal peptides were conjugated to KLH withglutaraldehyde, leaving a free C-terminus. Internal peptides wereMAP-conjugated with a blocked N-terminus. Additional immunoreagents canalso be generated by immunizing rabbits with the bacterially expressedGST-fusion proteins containing the cytoplasmic domains of each novelSTK.

The various immune sera are first tested for reactivity and selectivityto recombinant protein, prior to testing for endogenous sources.

Western Blots

Proteins in SDS PAGE are transferred to immobilon membrane. The washingbuffer is PBST (standard phosphate-buffered saline pH 7.4+0.1% triton x100). Blocking and antibody incubation buffer is PBST+5% milk. Antibodydilutions varied from 1:1000 to 1:2000.

Results

Three SULU1 antisera (against both 539A (SEQ ID NO: 79) and 540A (SEQ IDNO: 78)) and two SULU3 antisera (542A) (SEQ ID NO: 81) reactedspecifically with the peptide antigens. Antisera binding was competablewith peptide. Experiments with extracts from cells transfected withepitope-tagged SULU1 and SULU3 genes are underway.

Antisera against the PAK4 C-terminal peptide 554A (SEQ ID NO: 82)reacted with purified Gst-PAK4 and detected a protein of the correctmolecular weight from tissue culture cells. Specific immunoprecipitationexperiments are ongoing to determine the reactivity with native protein.

Similar immunization and antisera testing experiments are underway foreach of the other novel STE20-kinases.

STE20-related protein kinase peptide immunogens and their specificity inrecognizing endogenous protein by Western blots or immunoprecipitations.Pro- Aa tein Sequence positions Conj West. IP STLK2 EKFQKCSADESP 405-416KLH Y Y (SEQ ID No: 111) STLK4 SISNSELFPTTDPVGT 252-267 KLH Y Y (SEQ IDNO: 112) SULU1 LDFPKEDYR 890-898 KLH Y Y (SEQ ID NO: 113) SULU1HGDPRPEPRPTQ 409-420 KLH Y Y (SEQ ID NO: 114) SULU3 PSTNRAGSLKDPEC  2-14KLH N ND (SEQ ID NO: 115) SULU3 DPRTRASDPQSPPQVSRH 411-429 KLH ND ND K(SEQ ID NO: 116) PAK4 CLVPLIQLYRKQTSTC 666-680 KLH ND Y (SEQ ID NO: 117)PAK5 PLMRQNRTR 390-398 KLH Y Y (SEQ ID NO: 118) PAK5 SGDRRRAGPEKRPKSS148-163 KLH Y Y (SEQ ID NO: 119) PAK5 (C) RRKSLVGTPYWMAPE 471-485 KLH YND (SEQ ID NO: 120)ND = not done yet

STE20-related protein kinase GST fusion protein immunogens and theirspecificity in recognizing endogenous protein by Western blots orimmunoprecipitations. Protein domain Aa positions West. IP ZC1Coiled-coil/pro/B/C 350-867 Y Y ZC1 B 615-732 Y Y ZC2 Coiled-coil/pro/B348-762 ND ND ZC2 B 658-762 Y Y PAK4 Nterm 252-426 ND ND PAK4Kinase/Cterm 350-681 ND Y PAK5 A/Nterm  53-330 ND ND PAK5 A/Nterm 53-309 ND NDND = not done yet

The 50 kD STLK2 protein was expressed highly in several hematopoieticcell lines including Jurkat, pGL10, Ramos, A20, WEHI-231, K562, HEL andfreshly isolated thymocytes from C57/BL6 mice. High levels of STLK2expression were also detected in several tumor cell lines includingCalu6, Colo205, LS 180, MDAM231 and A549.

The 160 kD ZC1 protein was detected in Jurkat T cells, Colo205, HCT116,RIE-1, 293T, MDAMB231, and SK-MEL28.

The 170 kD ZC2 protein was detected in SK-Me128 and UACC-62.

Elevated levels of the 64 kD PAK5 protein were confirmed in the breastcancer cell lines MDA-231 and MCF-7, and in the lung cancer cell lineA549.

Example 5 Recombinant Expression and Biological Assays for STE20-RelatedProtein Kinases

Materials and Methods

Transient Expression of the Ste20-Related Kinases in Mammalian Cells

The pcDNA expression plasmids (10 μg DNA/100 mm plate) containing theSTE20-related kinase constructs are introduced into 293 cells withlipofectamine (Gibco BRL). After 72 hours, the cells are harvested in0.5 mL solubilization buffer (20 mM HEPES, pH 7.35, 150 mM NaCl, 10%glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA, 2 mMphenylmethylsulfonyl fluoride, 1 μg/mL aprotinin). Sample aliquots wereresolved by SDS polyacrylamide gel electrophoresis (PAGE) on 6%acrylamide/0.5% bis-acrylamide gels and electrophoretically transferredto nitrocellulose. Non-specific binding was blocked by preincubatingblots in Blotto (phosphate buffered saline containing 5% w/v non-fatdried milk and 0.2% v/v nonidet P-40 (Sigma)), and recombinant proteinwas detected using the various anti-peptide or anti-GST-fusion specificantisera.

In Vitro Kinase Assays

Three days after transfection with the STE20-related kinase expressioncontructs, a 10 cm plate of 293 cells was washed with PBS andsolubilized on ice with 2 mL PBSTDS containing phosphatase inhibitors(10 mM NaHPO₄, pH 7.25, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate,0.1% SDS, 0.2% sodium azide, 1 mM NaF, 1 mM EGTA, 4 mM sodiumorthovanadate, 1% aprotinin, 5 μg/mL leupeptin). Cell debris was removedby centrifugation (12000×g, 15 min, 4° C.) and the lysate was preclearedby two successive incubations with 50 μL of a 1:1 slurry of protein Asepharose for 1 hour each. One-half mL of the cleared supernatant wasreacted with 10 μL of protein A purified kinase-specific antisera(generated from the GST fusion protein or antipeptide antisera) plus 50μL of a 1:1 slurry of protein A-sepharose for 2 hr at 4° C. The beadswere then washed 2 times in PBSTDS, and 2 times in HNTG (20 mM HEPES, pH7.5/150 mM NaCl, 0.1% Triton X-100, 10% glycerol).

The immunopurified kinases on sepharose beads were resuspended in 20 μLHNTG plus 30 mM MgCl₂, 10 mM MnCl₂, and 20 μCi [α³²P]ATP (3000 Ci/mmol).The kinase reactions were run for 30 min at room temperature, andstopped by addition of HNTG supplemented with 50 mM EDTA. The sampleswere washed 6 times in HNTG, boiled 5 min in SDS sample buffer andanalyzed by 6% SDS-PAGE followed by autoradiography. Phosphoamino acidanalysis was performed by standard 2D methods on ³²P-labeled bandsexcised from the SDS-PAGE gel.

Similar assays were performed on bacterially expressed GST-fusionconstructs of the kinases.

ZC1 Assay buffer: 20 mM Tris pH 7.4, 200 mM NaCl, 0.5 mM DTT, 3 mMMgCl2, 0.3 mM MnCl2, 100 μM ³²PγATP.

Substrates: myelin basic protein (MBP) at 0.28 mg/mL and phosphorylatedZC1 peptide RTVGRRNTFIGT-PPYWMAPE (SEQ ID NO:121) at 17 μM (boldunderlined residue shows site of phosphorylation).

At higher concentrations of MgCl₂ (3 mM), the activity of ZC1 (bothfull-length and recombinant kinase domain) is up to 10-fold greatertowards exogenous substrate MBP. In contrast, the autophosphorylationand the phosphorylation of the activation loop peptide substrate areboth inhibited. Mn++ does not inhibit the autophosphorylation and thepeptide phosphorylation by the truncated kinase domain form. However,both the MBP phosphorylation, Mn++-preferring activity AND theautophosphorylating, Mg++-preferring activity are eliminated withmutation of the ATP-binding lysine in ZC1 (Lys54Ala) indicating thatboth activities are attributable to the ZC1 kinase domain.

SULU1 Assay buffer: This buffer is identical to that for ZC1, except for5 mM MgCl2. Under these conditions, other STE20 family members (PAK4,ZC1) were inhibited for autophosphorylation and required reducing the[Mn] to <0.3 mM for an efficient autophosphorylation reaction.

Substrates: MBP, phosvitin, or α-casein at 0.28 mg/mL.

PAK4, PAK5 Assay Buffer: 20 mM Hepes pH 7.2, 130 mM KCl, 10 mM MgCl2, 1mM NaF, 20 mM B-glycerolphosphate, 0.5 mM DTT, 50 μM ATP, 0.5 μCi³²PγATP.

Substrates: MBP at 0.28 mg/mL and peptide substrates derived from PAK5activation loop at 2.5 μM.

STLK2 Assay buffer: Similar to that described above, except for theinclusion of 5 mM MgCl₂, 5 mM MnCl₂ and 5 μCi ³²PγATP.

Transformation (PAK Experiments)

Low-passage NIH3T3 fibroblasts displaying normal morphology (flat,non-refractile cellular morphology), as well as low rates of spontaneoustransformation, were used in transformation assays. NIH3T3 cells weremaintained in Dulbecco's modified Eagle's medium supplemented with 10%(v/v) fetal calf serum, penicillin (100 U/mL) and streptomycin (100U/mL) and kept in an humidified incubator at 37° C. and 5% CO₂.

Cells were transfected with DNA-lipid complexes. As per manufacturerinstructions, lipofectamine was utilized to transfect NIH3T3 cells. Alltransfections were with equal amounts of plasmid DNA (DNA from theappropriate expression vector without insert was used to give equivalentamounts of DNA per transfection). 1 μg of activated allele of H-Ras wasco-transfected with increasing amounts of various alleles of PAK5.

Foci were scored after 3 weeks by fixing 10 min in 10% methanol, 10%acetic acid for 10 min, followed by staining with 0.4% (w/v) crystalviolet in 10% methanol for 10 min, and washing with deionized water anddrying at room temperature.

Transfections, Stimulations, and Luciferase Assays (ZC1 Experiments)

Cells (10⁷) were transiently transfected by electroporation using a GenePulser (Bio-Rad Labs) with the setting of 960 _F and 250 V. 20-40 hourslater, transfected cells (about 10⁵) were stimulated with variousstimuli. After a 6-hour stimulation, cells were lysed, and luciferaseactivities were measured using the MicroLumatPlus (EG&G Berthold). (J.Exp. Med. 183: 611-620, 1996, hereby incorporated by reference herein inits entirety including any drawings, tables, or figures.)

Results

Protein expression and kinase activity of novel STE20-related proteinkinases Endogenous Observed size Predicted In vitro Kinase KinaseProtein (kD) Size(kD) activity activity STLK2 50 46 y y STLK4 55 50 y NDZC1 160 140 y y ZC2 170 150 y y KHS2 ND 101 ND ND SULU1 119 105 y ySULU3 140 115 ND Y PAK4 80 75 y y PAK5 64 64 y yZC1: Regulation of Kinase activity

ZC1 is constitutively active as a full-length kinase when expressedeither in vitro (TNT rabbit reticulocyte system) or in NIH 3T3, 293T, orH1299 tissue culture cells. The endogenously expressed kinase is alsoactive when immunoprecipitated from carcinoma cell lines.

ZC1 Signaling Pathways

Using human leukemic T cell line Jurkat as a model system, the impact ofcotransfected wild-type ZC1 on the activation of two reporter genes,RE/AP-luciferase and NFκB luciferase, was examined. RE/AP is a compositein the IL-2 gene promoter containing both a NFκB-like site and an AP-1site.

Optimal activation of both RE/AP-luciferase and NFκB-luciferase reportergenes in Jurkat T cells requires signals generated from stimulation ofboth T cell receptor and the costimulator receptor CD28. Cotransfectionof wild-type ZC1 with either the RE/AP-luciferase or the NFκB-luciferasereporter results in the activation of RE/AP or NFκB when costimulatedwith the anti-T cell receptor monoclonal antibody or the pharmacologicalreagents PMA and ionomycin that bypass proximal T cell receptor. Noactivation was seen when costimulated with an anti-CD28 monoclonalantibody.

These results suggest that wild-type ZC1, when overexpressed, wasreplacing a CD28-specific signal to activate RE/AP or NFkB. Theseresults imply that ZC1 is involved in the CD28 signaling pathway. SinceNFκB is one of the major pathways also activated by the pro-inflammatorycytokine TNF-α signaling, it is also likely that ZC1 may be a componentin the TNF-α signaling pathways.

PAK5: Design of Specific Peptide Substrates

To aid in the development of in vitro kinase assays for screening smallmolecule libraries to identify specific inhibitors, the search forspecific peptide substrates for PAK5 was undertaken.

The rationale used to design such peptides is based on the hypothesisthat upon binding activated small G protein, PAK5 undergoes aconformational change that results in derepression of its kinaseactivity followed by autophosphorylation on the activation loopresulting in a fully active kinase. The site of autophosphorylation forrelated family members has been identified by biochemical and/or geneticmeans (e.g. Wu, C, et al. J. Biol. Chem 270: 15984-15992 andSzczepanowska, et al. Proc. Natl. Acad. Sci 94, 8503-8508, 1997).Specific peptide substrates for PAK5 were designed from the sequence ofthe activation loop of this kinase.

An activation loop PAK5 peptide phosphorylated on the Thr residue of theTPY motif served as a high-affinity substrate for PAK5.

PAK5 Activation Loop Peptides as Kinase Substrates Pep- tide SEQ Ki-sub- # Kinase Sequence Aa ID nase strate 1 PAK5 (C)RRKSLVGTPYWMA 471-485120 PAK5 yes PE 2 PAK5 (C)RRKSLVG T PYWMA 471-485 120 PAK5 yes PE 3 PAK5(C)RRK S LVGTPYWMA 471-485 120 PAK5 no PE 4 KHS1 KRKSFIGTPYWMAPE 171-185122 PAK5 yes 5 STLK2 KRNTFVGTPFWMA 175-189 123 PAK5 poor PE 6 SULU1PANSFVGTPYWMAPE 174-188 124 PAK5 poor 7 ZC1 RRNTFIGTPYWMAPE 184-198 125PAK5 poor 8 ZC1 RRNTFIG T PYWMAPE 184-198 126 PAK5 poor 9 STLK4RNKVRKTFVGTPCWM 66-83 127 PAK5 poor APE 10 PAK5 (C)RRKSLVG T PYWMA471-485 120 PAK4 yes PENote: underlined/bold reside was phosphorylated

Peptide # Kinase Notes 1 PAK5 Equally well as MBB 2 PAK5 High Km forPAK5 (1-10 μM) 3 PAK5 S is the site of phosphorylation 4 KHS1 Similar topeptide 1 5 STLK2 6 SULU1 7 ZC1 8 ZC1 Better than 7 9 STLK4 10 PAK5 SameKm as phosph. by PAK5PAK5: Transformation

Transformation of low-passage NIH3T3 cells by ras in the presence orabsence of various alleles of PAK5 showed that the dominant negative,kinase-dead allele of PAK5 was able to block ras transformation ofNIH3T3 cells. Thus, PAK5 activity is required for ras transformation ofNIH3T3 cells. Inhibition of PAK5 activity may have therapeutic value asan anti-proliferative agent for treating cancer.

PAK4 and PAK5: Interaction With Cdc42

PAK 4 interacts with CDC42 small G-protein but not Rac, RhoA, or Ras asdetermined by co-transfection of recombinant genes and detection bykinase assays. PAK5 also interacts with Cdc42. Coding sequences ofactivated alleles of small G proteins (ras, Cdc42, Rac, Rho) tagged witha Myc epitope were transiently expressed in 293T cells, various allelesof 35S-labeled PAK5 tagged with HA epitope were expressed in vitro withthe reticulocyte (TNT) system.

Example 6 Chromosomal Localization of Ste20-Related Protein Kinases

Materials And Methods

STE20 protein kinases STLK3, STLK4, ZC1, ZC2, ZC3, KHS2, SULU1, PAK4,and PAK5 were mapped using the GeneBridge 4 Radiation Hybrid Panel,RH02.05 (Research Genetics). The GeneBridge 4 Panel consists of 91hybrid panel samples, in addition to one human positive control (HFL),and one hamster negative control (A23). The standard reaction conditionsused to test and conduct PCR reactions using the GeneBridge 4 Panel areavailable from Research Genetics.

Oligonucleotide sequences (all 5′ to 3′) used for PCR mapping were:STLK3: CTCCCATTTCCTAGCAAAATCA, (SEQ ID NO: 128) AGAGGCAGTATTGTCAGATGTA(SEQ ID NO: 129) STLK4: CCACACATGCGTATCTCTGTTG, (SEQ ID NO: 130)TTGCTAGAATTCACATCAGGTACA (SEQ ID NO: 131) ZC1: ATCCCTGGATCACACTGCTTCT,(SEQ ID NO: 132) CAAGGTGTTCTTTGCCTCTGTT (SEQ ID NO: 133) ZC2:AGATGGACTGTACTGGGAGGG, (SEQ ID NO: 134) AGAAGAGCACTTGGCACTTATC (SEQ IDNO: 135) ZC3: CATCATGAACTGGTGACGGG, (SEQ ID NO: 136)CCAGTGAAATCAAACCAGTAAAA (SEQ ID NO: 137) SULU1:CAAAACCTGGCCGTCTCTTCTATT, (SEQ ID NO: 138) ATTTGTGCTACTGGGATTCTGTG (SEQID NO: 139) KHS2: GAATAGCGGTACCATGATAGAATA, (SEQ ID NO: 140)TACCAAAAAGAGCCAAAAGTGTG (SEQ ID NO: 141) PAK4: CTCAGTATTCTCTCCAAAGATTG,(SEQ ID NO: 142) GATGTTCTCTCCATTCTGTAAAG (SEQ ID NO: 143) PAK5:CATCACTGGAAGTCTGCAGTG, (SEQ ID NO: 144) CAGGTGCAGTAGTCATTTGC (SEQ ID NO:145)

Positive reactions were assigned a score of “1”, negative reactions areassigned a score of “0”, and ambiguous reactions are assigned a score of“2”. Results were submitted to the Whitehead Institute(www@genome.wi.mit.edu) for position analysis. Chromosomal localizationsfor ZC4, SULU3, STLK2, STLK5 and STLK6 were available publicly (forexample, from Unigene). The chromosomal locations of GEK2 and STLK7 havenot been determined. STLK2_h Xq25-27.1 (Public) STLK3 2q31.3 (Sugen)STLK4_h 3p22.3-p22.2 (Sugen) STLK5_h 17q23.2-24.2 (Public) STLK6_h2q32.2-q33.3 (Public) STLK7_h NA ZC1_h 2p11.2 (Sugen) ZC2_h3q26.31-3q26.32 (Sugen) ZC3_h 17p13.2-13.3 (Sugen) ZC4_h Xq22 (Public)KHS2_h 2p22-2p22.2 (Sugen) SULU1_h 12q24.21 (Sugen) SULU3_h 17p11.2(Public) GEK2_h NA PAK4_h 15q14 (Sugen) PAK5_h 19q13.2-q13.3 (Sugen)

Many of the STE 20 kinases were mapped to regions associated withvarious human cancers, as shown below.

The regions were also cross-checked with the Mendalian Inheritance inMan database, which tracks genetic information for many human diseases,including cancer. References for association of the mapped sites withchromosomal abnormalities found in human cancer can be found in:Knuutila, et al., Am J Pathol, 1998, 152: 1107-1123, hereby incorporatedherein be reference in its entirety including any figures, tables, ordrawings. Association of these mapped regions with other diseases isdocumented in the Online Mendalian Inheritance in Man (OMIM).

STLK2_h, Xq25-27.1, (Public)

-   Osteosarcoma, Xq25-qter, 2 of 31.-   Lymphoproliferative syndrome, X-linked (OMIM No. 308240)-   human STLK3, 2q31.3, (Sugen)-   Squamous cell carcinoma of Head and Neck, 3 of 30.    STLK4_h, 3p22.3-p22.2, (Sugen)-   Mantle cell lymphoma 3p14-p22 1 of 27-   Squamous cell carcinoma of Head and Neck 3p22-p24 1 of 14-   Cardiomyopathy, dilated (OMIM 601154)    STLK5_h, 17q23.2-24.2, (Public)-   Cervical cancer, 17q, 1 of 30-   Gastroesophageal junction adenocarcinoma xenograft, 17q, 1 of 5-   Breast carcinoma, 17q12-qter, 1 of 16-   Bladder carcinoma, 17q22-q23, 1 of 14-   Breast carcinoma, 17q22-q25, 8 of 101-   Non-small cell lung cancer, 17q24-q25, 6 of 50-   Testis, 17q24-qter, 2 of 11-   Malignant peripheral nerve sheath tumors, 17q24-qter, 5 of 7-   Alzheimer disease, susceptibility to (OMIM 106180)    STLK6_h, 2q32.2-q33.3, (Public)-   Non-small cell lung cancer, 2q31-q32, 1 of 50-   Squamous cell carcinoma of Head and Neck, 2q31-q33, 3 of 30-   Small cell lung cancer, 2q32-q35, 1 of 22    ZC1_h, 2p11.2, (Sugen)-   non-small cell lung cancer, 2pter-q13, 1 of 10-   non-small cell lung cancer, 2pter-q21, 1 of 10-   Pulmonary alveolar proteinosis, congenital (OMIM 178640).    ZC2_h, 3q26.31-3q26.32, (Sugen)-   Non-small cell lung cancer, 3q26.1-q26.3, 26 of 103-   Cervical cancer, 3q26.1-q27, 4 of 30-   Small cell lung cancer, 3q26.3-qter, 3 of 35-   Squamous cell carcinoma of Head and Neck, 3q26.3-qter, 3 of 13-   Marginal zone B-cell lymphoma, 3q26-q27, 1 of 25-   Parosteal osteosarcoma, 3q26-q28, 1 of 1-   Gastrointestinal stromal tumor, 3q26-q29, 1 of 16-   Mantle cell lymphoma, 3q26-q29, 1 of 5    ZC3_h 17p13.2-13.3 (Sugen)-   Malignant fibrous histiocytoma of soft tissue, 17p, 2 of 58-   Leiomyosarcoma, 17p, 7 of 29-   Non-small cell lung cancer, 17p, 1 of 50    ZC4_h, Xq22, (Public)-   Diffuse large cell lymphoma, Xq22-ter, 1 of 32-   Deafness, X-linked 1, progressive. (OMIM 304700).    KHS2_h, 2p22-2p22.2, (Sugen)-   Synovial sarcoma, 2p21-q14, 1_of_(—)67-   Follicular lymphoma, 2p22-p24, 1_of_(—)46-   Colorectal cancer, hereditary, nonpolyposis, type 1, Ovarian cancer    (MSH2, COCA1, FCC1). (OMIM 120435).    SULU1_h, 12q24.21 (Sugen)-   Neuroglial tumors, 12q22-qter, 1_of_(—)15-   Gastroesophageal junction adenocarcinoma, 12q23-qter, 1 of 5.-   Non-small cell lung cancer, 12q24.1-24.3, 2 of 50.    SULU3_h 17p11.2 (Public)-   Malignant fibrous histiocytoma of soft tissue, 17p, 2_of_(—)58-   Leiomyosarcoma, 17p, 7_of_(—)29-   non-small cell lung cancer, 17p, 1_of_(—)50-   Diffuse large cell lymphoma, 17p 11.2, 1_of_(—)32-   Osteosarcoma, 17p11.2-p12, 4_of_(—)31    PAK4_h: 15q14 (Sugen)-   Schizophrenia, (OMIM 118511).    PAK5_h: 19q13.2-q13.3 (Sugen)-   Follicular lymphoma, 19q13, 1 of 46*,-   Mantle cell lymphoma, 19q13, 1 of 5-   Hepatocellular carcinoma, 19q13.1, 2 of 50-   Small cell lung cancer, 19q13.1, 10 of 35-   Breast carcinoma, 19q13.1-qter, 1 of 33-   cervical cancer, 19q13.1-qter, 1 of 30-   Testis, 19q13.1-qter, 1 of 11-   Chondrosarcoma, 19q13.2, 1 of 29-   Malignant fibrous histiocytoma of soft tissue, 19q13.2-qter, 2 of 58-   Non-small cell lung cancer, 19qcen-q13.3, 6 of 104

Example 7 Demonstration of Gene Amplification By Southern Blotting

Materials and Methods

Nylon membranes were purchased from Boehringer Mannheim. Denaturingsolution contains 0.4 M NaOH and 0.6 M NaCl. Neutralization solutioncontains 0.5 M Tris-HCL, pH 7.5 and 1.5 M NaCl. Hybridization solutioncontains 50% formamide, 6×SSPE, 2.5× Denhardt's solution, 0.2 mg/mLdenatured salmon DNA, 0.1 mg/mL yeast tRNA, and 0.2% sodium dodecylsulfate. Restriction enzymes were purchased from Boehringer Mannheim.Radiolabeled probes were prepared using the Prime-it II kit byStratagene. The beta actin DNA fragment used for a probe template waspurchased from Clontech.

Genomic DNA was isolated from 20 different tumor cell lines: MCF-7,MDA-MB-231, Calu-6, A549, HCT-15, HT-29, Colo 205, LS-180, DLD-1,HCT-116, PC3, CAPAN-2, MIA-PaCa-2, PANC-1, AsPc-1, BxPC-3, OVCAR-3,SKOV3, SW 626 and PA-1, and from two normal cell lines: human mammaryepithelial cells and human umbilical vein endothelial cells.

A 10 μg aliquot of each genomic DNA sample was digested with EcoR Irestriction enzyme and a separate 10 μg sample was digested with HindIII restriction enzyme. The restriction-digested DNA samples were loadedonto a 0.7% agarose gel and, following electrophoretic separation, theDNA was capillary-transferred to a nylon membrane by standard methods(Sambrook, J. et al (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory).

PAK5 Amplicon:

A 600 base pair fragment (EcoR I-Sac I) of the PAK5 gene was used as atemplate for a radiolabeled DNA probe which was hybridized to the blotsat 42° C. for 48 hours in hybridization solution using standard methods(supra). The blots were exposed to a phosphorimager screen for 4 days,then scanned and analyzed using a Molecular Dynamics Storm 840phosphorimager. The relative mass and gene copy number values of thePAK5 DNA fragments were calculated from the band density valuesobtained. The blots were re-hybridized with a radiolabeled probe copiedfrom a fragment of human beta actin DNA and developed as above toconfirm the sample mass loading equivalency.

Results

The PAK5 gene was determined to exhibit 3-fold amplification compared tothe normal DNA copy number in PANC-1 (pancreatic epithelioid carcinoma)and OVCAR-3 (ovarian adenocarcinoma) human cell lines, and approximately2 times the normal copy number in the BxPC-3 (primary pancreaticadenocarcinoma) human cell line.

Similar Southern analyses can be performed for other STE20 kinases.

Example 8 Detection Of Protein-Protein Interaction Through Phage Display

Materials And Methods

Phage display provides a method for isolating molecular interactionsbased on affinity for a desired bait. cDNA fragments cloned as fusionsto phage coat proteins are displayed on the surface of the phage.Phage(s) interacting with a bait are enriched by affinity purificationand the insert DNA from individual clones is analyzed.

T7 Phage Display Libraries

All libraries were constructed in the T7Select1-1b vector (Novagen)according to the manufacturer's directions.

Bait Presentation

Protein domains to be used as baits were generated as C-terminal fusionsto GST and expressed in E. Coli. Peptides were chemically synthesizedand biotinylated at the N-terminus using a long chain spacer biotinreagent.

Selection

Aliquots of refreshed libraries (10¹⁰-10¹² pfu) supplemented with PanMixand a cocktail of E. coli inhibitors (Sigma P-8465) were incubated for1-2 hrs at room temperature with the immobilized baits. Unbound phagewas extensively washed (at least 4 times) with wash buffer.

After 3-4 rounds of selection, bound phage was eluted in 100 μL of 1%SDS and plated on agarose plates to obtain single plaques.

Identification of Insert DNAs

Individual plaques were picked into 25 μL of 10 mM EDTA and the phagewas disrupted by heating at 70° C. for 10 min. 2 μL of the disruptedphage were added to 50 μL PCR reaction mix. The insert DNA was amplifiedby 35 rounds of thermal cycling (94° C., 50 sec; 50° C., 1 min; 72° C.,1 min).

Composition of Buffer

-   10× PanMix-   5% Triton X100-   10% non-fat dry milk (Carnation)-   10 mM EGTA-   250 mM NaF-   250 μg/mL Heparin (sigma)-   250 μg/mL sheared, boiled salmon sperm DNA (sigma)-   0.05% Na azide-   Prepared in PBS    Wash Buffer-   PBS supplemented with:-   0.5% NP-40-   25 μl g/mL heparin    PCR Reaction Mix-   1.0 mL 10×PCR buffer (Perkin-Elmer, with 15 mM Mg)-   0.2 mL each dNTPs (10 mM stock)-   0.1 mLT7UP primer (15 pmol/μL) GGAGCTGTCGTATTCCAGTC-   0.1 mLT7DN primer (15 pmol/μL) AACCCCTCAAGACCCGTTTAG-   0.2 mL25 mM MgCl₂ or MgSO₄ to compensate for EDTA-   Q.S. to 10 mL with distilled water-   Add 1 unit of Taq polymerase per 50 μL reaction    PCR Reaction Mix-   1.0 mL 10×PCR buffer (Perkin-Elmer, with 15 mM Mg)-   0.2 mL each dNTPs (10 mM stock)-   0.1 mLT7UP primer (15 pmol/μL) GGAGCTGTCGTATTCCAGTC(SEQ ID NO:146)-   0.1 mLT7DN primer (15 pmol/μL) AACCCCTCAAGACCCGTTTAG (SEQ ID NO:147)-   0.2 mL 25 mM MgCl₂ or MgSO₄ to compensate for EDTA-   Q.S. to 10 mL with distilled water-   Add 1 unit of Taq polymerase per 50 μL reaction    LIBRARY: T7 Select1-H441

Results Phage display baits and interactors Sequence Patent CDNA RangeBait Domain Aa SEQ ID library Interactor & SEQ ID SULU1 Coiled-coil2752-898 22 H441 GEK2 cc dom (1) 677-820 SEQ #26 SULU3 Coiled-coil2755-898 23 H441 SLK isoform M83780SULU1 cc1 also interacted to a lesser extent with the coiled-coil domainof an SLK isoform.

The phage display data suggest potential interactions of SULU3 with SLKand SULU1 with GEK2 through their coiled-coil domains. Therefore twomembers of the SULU subfamily of STE20 kinases interact with two membersof a separate STE20 family, the prototype being SLK.

These results suggest a specificity in the interaction, and imply thatthese STE20 kinases may interact with each other through homo- andhetero-dimerization. Alternatively SULU-related kinases could actimmediately up- or down-stream of the SLK-related kinases in a signalingcascade.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art which are encompassed within the spirit ofthe invention are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed.

In particular, although some formulations described herein have beenidentified by the excipients added to the formulations, the invention ismeant to also cover the final formulation formed by the combination ofthese excipients. Specifically, the invention includes formulations inwhich one to all of the added excipients undergo a reaction duringformulation and are no longer present in the final formulation, or arepresent in modified forms.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

Other embodiments are within the following claims.

1. An antibody or an antigen-binding fragment thereof having specificbinding affinity to an epitope of a PAK5 kinase polypeptide.
 2. Anantibody or an antigen-binding fragment thereof according to claim 1,wherein said antibody is a monoclonal antibody.
 3. An antibody or anantigen-binding fragment thereof according to claim 1, wherein saidpolypeptide comprises the amino acid sequence set forth in SEQ ID NO:30.
 4. An antibody or an antigen-binding fragment thereof according toclaim 3, wherein said antibody is a monoclonal antibody.
 5. A hybridomaproducing the antibody or an antigen-binding fragment thereof ofclaim
 1. 6. A hybridoma producing the antibody or an antigen-bindingfragment thereof of claim
 3. 7. A method of detecting a PAK5 kinasepolypeptide or a PAK5 kinase polypeptide domain fragment in a samplecomprising: (a) contacting said sample with the antibody orantigen-binding fragment thereof of claim 1 under conditions such thatimmunocomplexes form, and (b) detecting the presence of said antibody orantigen-binding fragment thereof bound to said PAK5 kinase polypeptide.8. A method of detecting a PAK5 kinase polypeptide or a PAK5 kinasepolypeptide domain fragment in a sample comprising: (a) contacting saidsample with the antibody or antigen-binding fragment thereof of claim 3under conditions such that immunocomplexes form, and (b) detecting thepresence of said antibody or antigen-binding fragment thereof bound tosaid PAK5 kinase polypeptide.
 9. A PAK5 detection kit comprising: (a) afirst container means containing the antibody or antigen-bindingfragment thereof of claim 1 and (b) a second container means containinga labeled binding partner of said antibody or antigen-binding fragmentthereof.
 10. The PAK5 detection kit of claim 9 further comprising atleast one additional container means comprising one or more of the groupconsisting of wash reagents and reagents capable of detecting thepresence of bound antibodies.
 11. A PAK5 detection kit comprising: (a) afirst container means containing the antibody or antigen-bindingfragment thereof of claim 3 and (b) a second container means containinga labeled binding partner of said antibody or antigen-binding fragmentthereof.
 12. The PAK5 detection kit of claim 11 further comprising atleast one additional container means comprising one or more of the groupconsisting of wash reagents and reagents capable of detecting thepresence of bound antibodies.